Peptide chemically modified with polyethylene glycol

ABSTRACT

It is intended to provide a peptide chemically modified with PEG which contains a sequence consisting of 18 amino acids and having a specific structure made up of four planes, i.e., two hydrophobic planes and two hydrophilic planes alternately arranged in an alpha-helix structural model; a complex of the above peptide with a peptide-binding substance; a carrier modified with the peptide chemically modified with PEG as described above; a process for producing the same; and a method of delivering a substance bonded to a carrier modified with the peptide chemically modified with PEG or enclosed therein. The peptide chemically modified with PEG as described above has a high safety and can be easily formulated into a complex with a peptide-binding substance (i.e., having favorable handling properties). The resultant complex has a high solubility and shows an excellent introduction selectivity of the peptide-binding substance into cells. Thus, it is available as a vector achieving a high introduction efficiency without lowering the specific activity by the chemical modification with PEG.

TECHNICAL FIELD

This invention relates to a peptide which has been chemically modifiedwith polyethylene glycol (hereinafter abbreviated as “PEG”) (hereinafterabbreviated as “peptide chemically modified with PEG”), which peptideincludes a sequence of 18 amino acids which is constituted fromalternately arranged two hydrophobic sides and two hydrophilic sides ina-helix structural model depicted by Edmundson wheel plots, wherein atleast one of the two hydrophilic sides is a positively charged side.This invention also relates to its production method.

This invention also relates to a complex of a peptide chemicallymodified with PEG and a substance which binds to the peptide, and itsproduction method.

This invention also relates to a carrier modified with a peptidechemically modified with PEG, and its production method.

This invention also relates to a method for delivering a substance tothe interior of a cell, wherein the substance is bonded to orincorporated in the carrier which has been modified with the peptidechemically modified with PEG.

BACKGROUND ART

Entire genome sequences are being determined in an increasing number ofpathogenic microorganisms, pathogenic viruses, and human, and variousattempts are being made to treat various diseases by using the geneticinformation obtained. One such attempt is the treatment wherein a DNA,an RNA, or a derivative, a modification, or an analog thereof, namely, anucleic acid is administered to the interior of the body. Through suchadministration of the nucleic acid to the body, these treatments attemptto treat the disease by increasing or decreasing the amount ofparticular gene expressed or the extent of the function of particularphysiologically active factor developed, or by producing thephysiologically active substance coded by the introduced nucleic acid.In most of such treatment, a vector (an agent for introduction) is usedas a means for increasing the efficiency of nucleic acid introductioninto the cell.

One typical vector is a viral vector, wherein the infectivity inherentto the virus is utilized. Examples of such viral vectors includeretrovirus vector and adenovirus vector, and exemplary applications ofsuch viral vector are introduction of adenosine deaminase gene which isan attempt to treat adenosine deaminase deficiency by gene therapy(Blaese, R. M. et al., Science, Vol. 270, 475 (1995)), and introductionof p53 gene for cancer treatment (Swisher, S. G. et al., J. Natl. CancerInst., Vol. 91, 763 (1999)). A viral vector, however, is associated witha considerable risk in terms of its safety despite its excellentefficiency in introducing the nucleic acid to the cell. To be morespecific, a viral vector is known to suffer from the risk of emergenceof the wild type virus (pathogenic virus) and the risk of inducing aserious immune response by its high antigenicity. In addition, theprocess of production of a viral vector is an extremely complicated, andproduction of such viral vector in a commercial scale is generallydifficult.

Another typical vector is a liposome vector. This vector utilizes thephenomenon that a charged liposome becomes attached to the cell and theliposome then becomes incorporated in the cell, and the liposome vectorsknown in the art include liposomes having a nucleic acid incorporatedtherein, and the liposomes aggregated around a nucleic acid to form amass. Exemplary applications of the liposome having a nucleic acidincorporated therein include introduction of interferon β gene fortreating brain tumor (M. Mizuno et al., Cancer Res., Vol. 50, 7826(1990)), and exemplary uses of the method wherein a mass is formed byattaching liposomes around the nucleic acid include introduction of geneinto a cultivated cell, which is frequently conducted in cellengineering experiment (Felgner, P. L. et al., Proc. Natl. Acad. Sci.U.S.A., Vol. 84, 7413 (1987)). When the liposome vector is the onemainly comprising a natural phospholipid, the liposome vector is by farsuperior to the viral vector in view of safety. Such liposome vector,however, suffer from the problems of the complicated process of vectorproduction, the complicated process of producing the complex of thevector and the nucleic acid, and the low efficiency in introducing thenucleic acid in the cell. When the liposome is constituted from asynthetic lipid, efficiency of nucleic acid introduction into the celland handling convenience will be improved. Such liposome vector,however, suffers toxicity development. Also, both liposome vectors needfurther improvements in the drug preparation because of the poorstorability of the complex of the liposome and the nucleic acid.

Another exemplary vector is a peptide vector, and an example of suchpeptide vector is polylysine and its modified form. This vector utilizesthe nature that a positively charged peptide tends to electrostaticallybind to the negatively charged nucleic acid, and also to a cell. It hasbeen revealed from various studies that, when the polylysine is usedalone as a peptide vector, the oligonucleotide which has been covalentlybonded to the polylysine is introduced in the cell (Lemaitre, M. et al.,Proc. Natl. Acad. Sci. U.S.A., Vol. 84, 648 (1987)). However, it hasalso been revealed that modification of the polylysine with a sugar, aglycoprotein, a phospholipid, or the like is required to substantiallyintroduce the oligonucleotide or the plasmid that has beenelectrostatically bonded to the polylysine in a cell (Wu, G. Y. et al.,J. Biol. Chem., Vol. 262, 4429 (1987), Zhou, X. et al., Biochim. Biopys.Acta, Vol. 1065, 8 (1991), Liang, W. W. et al., Biochim. Biopys. Acta,Vol. 1279, 227 (1996)). It should also be noted that the unmodifiedpolylysine exhibits significant toxicity when administered into the bodyof an animal.

Another exemplary peptide vector is an amphipathic basic peptide havingα-helix structure, which has been shown to be a peptide usable alone asa vector for the nucleic acid since it exhibits high efficiency inintroducing the nucleic acid due to its structural characters (Niidome,T. et al., J. Biol. Chem., Vol. 272, 15307 (1997)).

This peptide, however, suffers from the drawback that, in the α-helixstructural model by Edmundson wheel plots, it exhibits typical two sidedstructure constituted from the hydrophobic side and the charged side(hydrophilic side), and when the proportion of the hydrophobic side isincreased in order to maintain the ability of introducing the nucleicacid into the cell, the solubility of the peptide in water is decreased.

In this respect, if the proportion of the hydrophobic side were reducedin order to improve the solubility in water, ability of introducing thenucleic acid into the cell would be compensated. In addition, theproportion of the hydrophilic side, which is the charged side, of thepeptide is small, and hydrophilicity of the peptide is lost once thenucleic acid has become electrostatically bound to the peptide, and thecomplex of the peptide and the nucleic acid becomes hard to be soluble.As a consequence, the peptide suffers from the drawback that aggregatesare liable to be formed, and such aggregate formation is a substantialproblem. Furthermore, the peptide will be highly toxic when it isadministered to the interior of the animal body due to the high tendencyof the aggregate formation in serum.

As described above, despite the excellent general handling convenienceof the peptide vector that it is capable of forming a complex merely bymixing with the nucleic acid, the peptide vector suffers from thedrawback that it has a high tendency of forming aggregates especially inblood, and in addition, that the complex of the peptide vector and thenucleic acid exhibits a low solubility, and hence, a high toxicity.Therefore, the peptide vector has drawbacks to practical use.

In addition, all of the above-described vectors had no selectivity forthe cell to which it is to be introduced, and the nucleic acid wasintroduced not only to the cell wherein the introduction of the nucleicacid was intended but also to the cell wherein the introduction of thenucleic acid was not at all intended. Such non-selective introductionhas been associated with the risk of developing side effects.

By the way, phosphatidyl serine and phosphatidyl ethanolamine areaminophospholipids which are constituents of the lipid bilayerconstituting the cell surface layer, and these aminophospholipids arephospholipids whose ratio of the content in the outer layer to thecontent in the inner layer of the lipid bilayer varies according to theconditions of the cell.

To be more specific, phosphatidyl serine and phosphatidyl ethanolamineare phospholipids whose content in the outer layer of the lipid bilayerof the cell membrane increases in relation to the content in the innerlayer when the cell receives some stimulus as typically found in thecell in the site where blood coagulation reaction is proceeding (Alan J.Schroit et al., Biochim. Biophys. Acta, Vol. 1071, 313 (1991)).Proportion of these phospholipids in the outer layer of the lipidbilayer is also believed to increase in the cells at the site whereinflammation or cell activation and/or injury, apoptosis, or otherso-called immunoresponsive reaction caused by immunocompetent cell hastaken place, the site where cells have become malignantly transformedthrough the progress of abnormal cell division, the site where the cellsconstituting the blood vessel have been injured by blood coagulation orby the progress of arterial sclerosis, the site where a cytotoxicreaction induced by active oxygen is in progress, and the site wherecell activation and/or cell injury by protease is in progress, and to bemore specific, in the injured, denatured, or activated cell, namely, inthe so-called abnormal cell. Phosphatidyl serine is also known to be aphospholipid which is found in the granule, and which becomestranslocated to the cell surface as a content of the granule in thecourse of degranulation in the cell (for example, mast cell or basophil)experiencing an allergic reaction (degranulation) caused by the bindingof an allergen to the IgE antibody on the cell surface (Martin, S. etal., Int. Arch. Allergy and Immunol., Vol. 123, 249 (2000)).

Recent studies report that, even in the case of a cell which has beenadministered with an apoptosis-inducing substance (for example, ananticancer drug) or a cell which has been irradiated with radiation, andeven if the cell had experienced signal transduction wherein p53 orother apoptosis-related gene had been involved, apoptosis does not takeplace if the cell is drug resistant (anticancer drug-resistant cancercell etc.), and the drug resistant cell survives after DNA repair, andin the course of such DNA repair, phosphatidyl serine is translocated tothe cell surface layer (Geske, F J. et al., Cell Death Differ., Vol. 8,182 (2001)).

Many studies have revealed that modification of a physiologically activepeptide or polypeptide with PEG is an effective way of promoting in vivofunctioning of the peptide or the polypeptide. Such modification withthe PEG presumably results in an increased hydrophilicity of the peptideor the polypeptide which in turn results in the reduced in vivainteraction with proteins as well as less likeliness of being recognizedby reticuloendothelial system, and hence, reduced possibility of beingcaptured by macrophage and the like. As a consequence, in vivo dynamicsis improved to enable maintenance of the in vivo physiological activity.Exemplary uses of the PEG modification include the case of PEG-ADA whichis adenosine deaminase (ADA) modified with PEG whose clinicaleffectiveness has been proven in the treatment of ADA deficiency(Hershfield M. S. et al., N. Engl. J. Med., 316, 589 (1987)) and thecase of the modification of the interferon (IFN) with PEG which hasresulted in the enhancement of the in vivo antiviral action (Perry C Met al., Drugs, 61, 2263 (2001)).

Usefulness of the PEG modification has been revealed not only for thephysiologically active peptide and polypeptide but also for a drugcarrier used for the purpose of drug delivery (Maruyama, K., NihonRinsho, 80, 632 (1998)). For example, liposome modification with PEG hasbeen revealed to be effective in extending the life in the body and alsoin reducing antigenicity due to the reduced recognizability by thereticuloendothelial system, as in the case of the PEG-modified peptideand polypeptide. Exemplary such uses of the modification of the drugcarrier with PEG include the case wherein a liposome having ananticancer drug doxorubicin incorporated therein is modified with PEG,the modification resulting in an increased drug effectivity compared tothe unmodified liposome (Sakakibara, T. et al., Cancer Res., 56, 3743(1996)).

Attempts have also been made to impart site targetability to thePEG-modified carrier in order to reduce the side effects and improve thedrug effectivity. An exemplary such attempt is the modification of aPEG-modified carrier further with a PEG-modified antibody (Maruyama K.et al., Biochim. Biophys. Acta, 1234, 74 (1995)).

Examples other than the liposome include PEG-modified polylysine used inthe introduction of a gene (Lee, M. et al., Mol. Ther., 4, 339 (2001)).

Despite the high toxicity of the polylysine, it has been known that thetoxicity of polylysine can be reduced by the modification with PEG.

While the PEG modification is a useful technique as described above, ithas the drawback that the peptide or the polypeptide after themodification exhibits reduced specific activity, which is caused by thePEG modification of the active center. For example, specific activity ofarginase has been shown to reduce to 65% when it is modified with PEG(Savoca, K. V. et al., Biochim. Biophys. Acta, 578, 47 (1979)).

Accordingly, usefulness of the PEG modification is expected to beimproved if PEG modification can be accomplished without reducing thespecific activity.

When a PEG-modified antibody is used as a means for imparting the sitetargetability to the PEG-modified carrier, there is a problem in that anantibody is susceptible to steric hindrance due to the PEG molecule andthe effect is less likely to be realized. Accordingly, development of amore effective means for imparting site targetability is awaited.

In view of such situation, an object of the present invention is toprovide a novel peptide chemically modified with PEG which is highlysafe; which can easily make a complex with a substance which binds tothe peptide (enjoying excellent handling convenience), the thus producedcomplex exhibits excellent solubility; which can serve a vector withhigh selective and efficient introduction of the substance which bindsto the peptide into a cell; and whose specific activity has not beencompensated by the chemical modification with the PEG; as well as itsproduction method.

Another object of the present invention is to provide a complex of thepeptide chemically modified with PEG and a substance which binds to thepeptide, and its production method.

A further object of the present invention is to provide a peptidechemically modified with PEG which can impart a site targetability to acarrier having a drug bound thereto or incorporated therein; a carriermodified with such peptide; and their production method.

A still further object of the present invention is to provide a methodfor delivering a substance bound to or incorporated in the carrier,which has been modified with the peptide chemically modified with PEG,into the cell.

DISCLOSURE OF THE INVENTION

The inventors of the present invention have made an intensive study, andfound that:

(1) a peptide which binds to the substance that binds to the peptide(the peptide used in the present invention and the peptide chemicallymodified with PEG of the present invention as will be described below)(herein abbreviated as the “peptide-binding substance”) and which hasaffinity for a certain phospholipid can be a vector for thepeptide-binding substance which exhibits high efficiency in introducingthe peptide-binding substance into a cell, low toxicity, and anexcellent solubility;

(2) a peptide which has a particular structural character defined by theamino acid sequence exhibits high localizability on a particularphospholipid, and hence, high selectivity in introducing thepeptide-binding substance into a particular cell;

(3) efficiency in introducing the peptide-binding substance into thecell can be improved by chemically modifying the peptide with PEG;

(4) the peptide chemically modified with PEG obtained by modifying thepeptide with PEG can be used as an element for imparting sitetargetability to a carrier having a drug bound thereto or incorporatedtherein; and

(5) the substance which has been bound to or incorporated in the carrierwhich is modified with the peptide chemically modified with PEG isefficiently delivered to a particular cell.

The present invention has been completed on the basis of such findings.

Accordingly, the present invention provides a peptide chemicallymodified with PEG according to (1) to (9) or (21), below; a complex ofthe peptide chemically modified with PEG and the peptide-bindingsubstance according to (10) to (19) or (24), below; a method forproducing the peptide or the complex according to (20), (22), or (23),below; a carrier which is modified with the peptide chemically modifiedwith PEG according to (25) or (27), below; a method for producing thecarrier according to (26), below; and a method for delivering asubstance according to (28), below.

The first aspect of the present invention is as follows:

(1) A peptide chemically modified with polyethylene glycol (PEG),including a sequence of 18 amino acids, wherein

-   -   said sequence of 18 amino acids is constituted from alternately        arranged two hydrophobic sides and two hydrophilic sides in        a-helix structural model depicted by Edmundson wheel plots,

one of said hydrophobic sides comprises 5 to 7 amino acids and 80 mole %or more of this side comprises hydrophobic amino acids,

one of said hydrophilic sides comprises 5 or 6 amino acids, and 80 mole% or more of this side comprises hydrophilic amino acids, and 50 mole %or more of this side comprises an amino acid selected from the groupconsisting of arginine and lysine,

the other of said hydrophobic sides comprises 2 to 4 hydrophobic aminoacids, and

the other of said hydrophilic sides comprises 3 to 5 amino acids and 80mole % or more of this side comprises hydrophilic amino acids.

(2) A peptide chemically modified with PEG according to (1) wherein saidpeptide comprises 20 or more amino acids in total; opposite ends of saidpeptide are N and C terminals; and any 18 consecutive amino acids insaid peptide excluding the amino acids at opposite ends constitutes saidsequence of 18 amino acids.

(3) A peptide chemically modified with PEG according to (1) or (2)wherein the amino acids at the N and C terminals are each a hydrophilicamino acid.

(4) A peptide chemically modified with PEG according to any one of (1)to (3) wherein said sequence of 18 amino acids is a sequence of any 18consecutive amino acids in the following amino acid sequence:X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-X32-X33-X34-X35-X36,provided that,

in each of “X4, X8, X11, X15, and X19”, “X8, X11, X15, X19, and X22”,“X11, X15, X19, X22, and X26”, “X15, X19, X22, X26, and X29”, and “X19,X22, X26, X29, and X33”, at least 4 amino acids out of the 5 amino acidsare a hydrophobic amino acid,

X3, X10, X12, X21, X28, and X30 are independently a hydrophobic aminoacid, a neutral hydrophilic amino acid, or a basic hydrophilic aminoacid,

in each of “X2, X5, X9, X13, and X16”, “X5, X9, X13, X16, and X20”, “X9,X13, X16, X20, and X23”, “X13, X16, X20, X23, and X27”, “X16, X20, X23,X27, and X31”, and “X20, X23, X27, X31, and X34”, at least 4 amino acidsout of the 5 amino acids are a neutral hydrophilic amino acid or a basichydrophilic amino acid, at least 3 amino acids of which being arginineor lysine,

X6, X17, X24, and X35 are independently a hydrophobic amino acid, and

X7, X14, X18, X25, X32, and X36 are independently a neutral hydrophilicamino acid or a basic hydrophilic amino acid.

(5) A peptide chemically modified with PEG according to any one of (1)to (4) wherein peptide moiety of said peptide chemically modified withPEG and including said sequence of 18 amino acids comprises thefollowing amino acid sequence:X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-X32-X33-X34-X35-X36-X37,provided that

X1 and X37 are a hydrophilic amino acid,

in each of “X4, X8, X11, X15, and X19”, “X8, X11, X15, X19, and X22”,“X11, X15, X19, X22, and X26”, “X15, X19, X22, X26, and X29”, and “X19,X22, X26, X29, and X33”, at least 4 amino acids out of the 5 amino acidsare a hydrophobic amino acid,

X3, X10, X12, X21, X28 and X30 are independently a hydrophobic aminoacid, a neutral hydrophilic amino acid, or a basic hydrophilic aminoacid,

in each of “X2, X5, X9, X13, and X16”, “X5, X9, X13, X16, and X20”, “X9,X13, X16, X20, and X23”, “X13, X16, X20, X23, and X27”, “X16, X20, X23,X27, and X31”, and “X20, X23, X27, X31, and X34”, at least 4 amino acidsout of the 5 amino acids are a neutral hydrophilic amino acid or a basichydrophilic amino acid, at least 3 amino acids of which being arginineor lysine,

X6, X17, X24, and X35 are independently a hydrophobic amino acid, and

X7, X14, X18, X25, X32, and X36 are independently a neutral hydrophilicamino acid or a basic hydrophilic amino acid; and wherein the sequenceof amino acids X2 to X36 may include deletion, addition, insertion, orsubstitution as long as at least 18 amino acids are conserved inconsecutive form.

(6) A peptide chemically modified with PEG according to (5) wherein X1to X37 are the following amino acids:

X1 is threonine,

X37 is serine,

X2, X5, X9, X20, X23, and X27 are independently arginine or lysine,

X3 and X21 are independently tyrosine, phenylalanine, serine, orarginine,

X4, X17, X22, and X35 are independently leucine,

X6, X15, X24, and X33 are independently leucine or isoleucine,

X7, X13, X25, and X31 are independently histidine or arginine,

X8 and X26 are independently proline,

X10 and X28 are independently serine, arginine, or leucine,

X11 and X29 are independently tryptophan or leucine,

X12 and X30 are independently valine, leucine, or serine,

X14 and X32 are independently glutamine, asparagine, or arginine,

X16 and X34 are independently alanine or arginine,

X18 is arginine, lysine, or serine,

X19 is leucine or threonine, and

X36 is arginine or serine; and wherein the sequence of amino acids X2 toX36 may include deletion, addition, insertion, or substitution as longas at least 18 amino acids are conserved in consecutive form.

(7) A peptide chemically modified with PEG according to any one of (1)to (6) wherein peptide moiety of said peptide chemically modified withPEG and including said sequence of 18 amino acids comprises any one ofthe amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 24.

(8) A peptide chemically modified with PEG according to any one of (1)to (6) wherein peptide moiety of said peptide chemically modified withPEG and including said sequence of 18 amino acids comprises the aminoacid sequence of SEQ ID NO: 16 or SEQ ID NO: 19.

(9) A peptide chemically modified with PEG according to any one of (1)to (8) wherein PEG moiety of said peptide chemically modified with PEGand including said sequence of 18 amino acids has a molecular weight ofabout 200 Da to about 100,000 Da.

The second aspect of the present invention is as follows:

(10) A complex comprising a peptide chemically modified with PEG andincluding a sequence of 18 amino acids, and a substance which binds tosaid peptide wherein

said sequence of 18 amino acids is constituted from alternately arrangedtwo hydrophobic sides and two hydrophilic sides in a-helix structuralmodel depicted by Edmundson wheel plots,

one of said hydrophobic sides comprises 5 to 7 amino acids and 80 mole %or more of this side comprises hydrophobic amino acids,

one of said hydrophilic sides comprises 5 or 6 amino acids, and 80 mole% or more of this side comprises hydrophilic amino acids, and 50 mole %or more of this side comprises an amino acid selected from the groupconsisting of arginine and lysine,

the other of said hydrophobic sides comprises 2 to 4 hydrophobic aminoacids, and

the other of said hydrophilic sides comprises 3 to 5 amino acids and 80mole % or more of this side comprises hydrophilic amino acids.

(11) A complex according to (10) wherein said peptide comprises 20 ormore amino acids in total; opposite ends of said peptide are N and Cterminals; and any 18 consecutive amino acids in said peptide excludingthe amino acids at opposite ends constitutes said sequence of 18 aminoacids.

(12) A complex according to (10) or (11) wherein the amino acids at theN and C terminals are each a hydrophilic amino acid.

(13) A complex according to any one of (10) to (12) wherein saidsequence of 18 amino acids is a sequence of any 18 consecutive aminoacids in the following amino acid sequence:X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-X32-X33-X34-X35-X36,provided that,

in each of “X4, X8, X11, X15, and X19”, “X8, X11, X15, X19, and X22”,“X11, X15, X19, X22, and X26”, “X15, X19, X22, X26, and X29”, and “X19,X22, X26, X29, and X33”, at least 4 amino acids out of the 5 amino acidsare a hydrophobic amino acid,

X3, X10, X12, X21, X28, and X30 are independently a hydrophobic aminoacid, a neutral hydrophilic amino acid, or a basic hydrophilic aminoacid,

in each of “X2, X5, X9, X13, and X16”, “X5, X9, X13, X16, and X20”, “X9,X13, X16, X20, and X23”, “X13, X16, X20, X23, and X27”, “X16, X20, X23,X27, and X31”, and “X20, X23, X27, X31, and X34”, at least 4 amino acidsout of the 5 amino acids are a neutral hydrophilic amino acid or a basichydrophilic amino acid, at least 3 amino acids of which being arginineor lysine,

X6, X17, X24, and X35 are independently a hydrophobic amino acid, and

X7, X14, X18, X25, X32, and X36 are independently a neutral hydrophilicamino acid or a basic hydrophilic amino acid.

(14) A complex according to any one of (10) to (13) wherein peptidemoiety of said peptide chemically modified with PEG and including saidsequence of 18 amino acids comprises the following amino acid sequence:X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-X32-X33-X34-X35-X36-X37,provided that

X1 and X37 are a hydrophilic amino acid,

in each of “X4, X8, X11, X15, and X19”, “X8, X11, X15, X19, and X22”,“X11, X15, X19, X22, and X26”, “X15, X19, X22, X26, and X29”, and “X19,X22, X26, X29, and X33”, at least 4 amino acids out of the 5 amino acidsare a hydrophobic amino acid,

X3, X10, X12, X21, X28 and X30 are independently a hydrophobic aminoacid, a neutral hydrophilic amino acid, or a basic hydrophilic aminoacid,

in each of “X2, X5, X9, X13, and X16”, “X5, X9, X13, X16, and X20”, “X9,X13, X16, X20, and X23”, “X13, X16, X20, X23, and X27”, “X16, X20, X23,X27, and X31”, and “X20, X23, X27, X31, and X34”, at least 4 amino acidsout of the 5 amino acids are a neutral hydrophilic amino acid or a basichydrophilic amino acid, at least 3 amino acids of which being arginineor lysine,

X6, X17, X24, and X35 are independently a hydrophobic amino acid, and

X7, X14, X18, X25, X32, and X36 are independently a neutral hydrophilicamino acid or a basic hydrophilic amino acid; and wherein the sequenceof amino acids X2 to X36 may include deletion, addition, insertion, orsubstitution as long as at least 18 amino acids are conserved inconsecutive form.

(15) A complex according to (14) wherein X1 to X37 are the followingamino acids:

X1 is threonine,

X37 is serine,

X2, X5, X9, X20, X23, and X27 are independently arginine or lysine,

X3 and X21 are independently tyrosine, phenylalanine, serine, orarginine,

X4, X17, X22, and X35 are independently leucine,

X6, X15, X24, and X33 are independently leucine or isoleucine,

X7, X13, X25, and X31 are independently histidine or arginine,

X8 and X26 are independently proline,

X10 and X28 are independently serine, arginine, or leucine,

X11 and X29 are independently tryptophan or leucine,

X12 and X30 are independently valine, leucine, or serine,

X14 and X32 are independently glutamine, asparagine, or arginine,

X16 and X34 are independently alanine or arginine,

X18 is arginine, lysine, or serine,

X19 is leucine or threonine, and

X36 is arginine or serine; and wherein the sequence of amino acids X2 toX36 may include deletion, addition, insertion, or substitution as longas at least 18 amino acids are conserved in consecutive form.

(16) A complex according to any one of (10) to (15) wherein peptidemoiety of said peptide chemically modified with PEG and including saidsequence of 18 amino acids comprises any one of the amino acid sequencesof SEQ ID NO: 1 to SEQ ID NO: 24.

(17) A complex according to any one of (10) to (15) wherein peptidemoiety of said peptide chemically modified with PEG and including saidsequence of 18 amino acids comprises the amino acid sequence of SEQ IDNO: 16 or SEQ ID NO: 19.

(18) A complex according to any one of (10) to (17) wherein saidsubstance which binds to the peptide is a nucleic acid.

(19) A complex according to any one of (10) to (18) wherein PEG moietyof said peptide chemically modified with PEG and including said sequenceof 18 amino acids has a molecular weight of about 200 Da to about100,000 Da.

(20) A method for producing the peptide chemically modified with PEG ofany one of (1) to (9) comprising the step of reacting a peptidecomprising said sequence of 18 amino acids with activated polyethyleneglycol.

(21) A peptide chemically modified with polyethylene glycol (PEG) whichis produced by the method of (20).

(22) A method for producing the complex of any one of (10) to (19)comprising the steps of

a) reacting a peptide comprising said sequence of 18 amino acids withactivated polyethylene glycol (PEG), and

b) reacting the peptide chemically modified with PEG that is obtained insaid a) with a substance which binds to said peptide.

(23) A method for producing the complex of any one of (10) to (19)comprising the steps of

a) reacting a peptide comprising said sequence of 18 amino acids with asubstance which binds to said peptide, and

b) reacting the reaction product of said peptide and said substancewhich binds to said peptide with activated polyethylene glycol (PEG).

(24) A complex of a peptide chemically modified with polyethylene glycol(PEG) and a substance which binds to said peptide, said complex beingproduced by the method of (22) or (23).

(25) A carrier which is modified with the peptide chemically modifiedwith PEG according to any one of (1) to (8).

(26) A method for producing the carrier of (25) which is modified withthe peptide chemically modified with PEG comprising the steps of

a) reacting a peptide comprising said sequence of 18 amino acids, or apeptide comprising said sequence of 18 amino acids and having cysteineattached to N or C terminal of the peptide, with activated PEG, and

b) reacting the reaction product of said a) with a carrier, orconstructing a carrier by using the reaction product of said a) as aconstituent.

(27) A carrier which is modified with the peptide chemically modifiedwith PEG, said carrier being produced by the method of (26).

(28) A method for delivering a substance to the interior of a cell, saidsubstance being bonded to or incorporated in the carrier of (25) thathas been modified with the peptide chemically modified with PEG.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(A), 1(B), and 1(C) are schematic views showing α-helixstructural model by Edmundson wheel plots. (In the figures, the aminoacids surrounded by square are hydrophobic amino acids, the amino acidssurrounded by circle are basic hydrophilic amino acids, and the aminoacids not surrounded by any of these are neutral hydrophilic aminoacids. The same also applies to other figures).

FIG. 2 is a view showing an exemplary four sided structure of thesequence of 18 amino acids in the peptide used in the present invention.

FIGS. 3(A) and 3(B) are views showing an exemplary four sided structureof the peptide used in the present invention with the amino acidsallocated to respective sides in a different manner.

FIG. 4(a) shows (C1), and FIG. 4(b) shows (C2). (C1) and (C2) are viewsshowing an exemplary four sided structure of the peptide used in thepresent invention with the amino acids allocated to respective sides ina different manner.

FIG. 5(A) and FIG. 5(B) are views showing an exemplary four sidedstructure of the peptide of the present invention including the sequenceof 18 amino acids.

FIG. 6(a) shows (C), and FIG. 6(b) shows (D). (C) and (D) are viewsshowing an exemplary four sided structure of the peptide of the presentinvention including the sequence of 18 amino acids.

FIG. 7(a) is a view showing mean residue ellipticity in CD spectroscopyunder SDS(−) conditions. FIG. 7(b) is a view showing mean residueellipticity in CD spectroscopy under SDS(+) conditions.

FIG. 8(a) is a view showing mean residue ellipticity in CD spectroscopyunder SDS(−) conditions. FIG. 8(b) is a view showing mean residueellipticity in CD spectroscopy under SDS(+) conditions.

FIG. 9(a) is a view showing mean residue ellipticity in CD spectroscopyunder SDS(−) conditions. FIG. 9(b) is a view showing mean residueellipticity in CD spectroscopy under SDS(+) conditions.

FIG. 10 is an electropherogram of the mixture of the peptide of SEQ IDNO: 1 and an oligonucleotide.

FIG. 11 is an electropherogram of the mixture of the peptide of SEQ IDNO: 1 and a plasmid.

FIG. 12 is a view showing how a plasmid was introduced in the cell inthe presence and absence of the peptide of SEQ ID NO: 1 by using theluciferase activity expressed by the plasmid as the index.

FIG. 13 is a view showing increase of GCV sensitivity when a plasmidincluding HSV-tk gene was introduced in a cell by using the peptide ofSEQ ID NO: 16.

FIG. 14 is a view showing the ability of the peptide of SEQ ID NO: 1 forintroducing a plasmid in a cell before and after storing the complex ofthe peptide with the plasmid at 4° C. by using the luciferase activityexpressed by the plasmid as the index.

FIG. 15 is an electropherogram of an oligonucleotide after treating themixture of the peptide of SEQ ID NO: 1 and the oligonucleotide with anuclease.

FIG. 16 is an electropherogram of a plasmid after treating the mixtureof the peptide of SEQ ID NO: 16 and the plasmid with a nuclease. Controlis the plasmid which has not been treated with the nuclease.

FIG. 17 is a view showing the specific affinity of the peptide of SEQ IDNO: 1 for phosphatidyl serine which has been measured by using Biacore2000.

FIG. 18 is a view showing the measurements obtained by using Biacore2000. The measurements indicate that the peptide of SEQ ID NO: 25 has noaffinity for either phosphatidyl serine or phosphatidyl choline.

FIG. 19 is a view showing the results of flow cytometry showing that,when a cell is stimulated for degranulation, phosphatidyl serine istranslocated to the surface of the cell.

FIG. 20 is a view showing that the peptide of SEQ ID NO: 16 introduces alarger amount of gene into the cell which has undergone degranulationwith the phosphatidyl serine translocated to its surface.

FIG. 21 is a view showing that the peptide of SEQ ID NO: 16 is capableof introducing a plasmid into the cancer cell that had been transplantedin a mouse, by using the luciferase activity expressed by the plasmid asthe index.

FIG. 22 is a view showing that survival period of a mouse can beextended by introducing the plasmid containing HSV-tk gene in the cancercell that had been transplanted in a mouse by using the peptide of SEQID NO: 16, and thereafter administering GCV.

FIG. 23 is a graph wherein gene introduction ability is compared betweenthe PEG-modified plasmid/peptide complex and the correspondingPEG-unmodified complex by using Vero cell.

FIG. 24 is a graph wherein gene introduction ability is compared betweenthe PEG-modified plasmid/peptide complex and the correspondingPEG-unmodified complex by using anaphylactic shock mice.

FIG. 25 shows the results of SDS-PAGE wherein PEG-modified peptide inthe PEG-modified plasmid/peptide complex was identified.

BEST MODE FOR CARRYING OUT THE INVENTION

Next, the present invention is described in further detail.

The peptide chemically modified with PEG of the present invention(according to the first aspect of the present invention) is a peptidechemically modified with PEG (hereinafter referred to as “the peptidechemically modified with PEG”) including an amino acid sequencecomprising 18 amino acids, wherein said sequence of 18 amino acids isconstituted from four sides comprising alternately arranged twohydrophobic sides (side A and side C) and two hydrophilic sides (side Band side D) in α-helix structural model depicted by Edmundson wheelplots (Edmundson, A. B. et al., Biophys. J., 7, 121 (1967)), and atleast one side (side B) of the two hydrophilic sides (side B and side D)is a positively charged side.

The peptide chemically modified with PEG according to the presentinvention is obtained by chemically modifying the peptide containing thesequence comprising 18 amino acids (hereinafter abbreviated as “thepeptide used in the present invention”) as will be described below, forexample, with activated PEG.

The method used for the chemical modification with PEG is not limited toany particular method, and exemplary methods include those using anactivated PEG and those using PEG and an activating agent.

The activated PEG used for the chemical modification is not particularlylimited as long as it is an activated PEG, and may comprise a straightchain or a branched chain structure. Examples of the activated PEGinclude mPEG-SPA (succinimidyl ester of methoxy poly(ethylene glycol)propionic acid manufactured by Shearwater) and other products ofShearwater such as mPEG-SBA (succinimidyl ester of mPEG butanoic acid),mPEG-SS (succinimidyl ester of mPEG succinate), mPEG-SCM (succinimidylester of carboxymethylated mPEG), mPEG-BTC (benzotriazole carbonatederivative of mPEG), mPEG-epoxide, mPEG-CDI(carbonyldiimidazole-activated mPEG), mPEG-NPC (p-nitrophenyl mPEGcarbonate), mPEG-aldehyde, mPEG-Isocyanate, mPEG-maleimide, andmPEG-OPSS (mPEG orthopyridyl disulfide). A PEG synthesized by a knownmethod is also useful.

The activated PEG used for the chemical modification may also be PEGhaving a phospholipid attached thereto, for example, MAL-mPEG-DSPE(maleimide modified and distearoylphosphatidylethanolamine modifiedmPEG), and NHS-mPEG-DSPE (N-hydroxysuccinimidyl carbonate modifiedmPEG-DSPE).

The average molecular weight of PEG may range from about 200 Da to about100,000 Da, and preferably about 1,000 Da to about 50,000 Da, and morepreferably about 2,000 Da to about 20,000 Da.

When the average molecular weight is within such range, the PEG-modifiedpeptide will retain the activity of the original peptide after thereaction between the activated PEG and the peptide, and the PEG-modifiedpeptide will be allowed to have favorable characters such as improvedsolubility, reduced antigenicity, and reduced toxicity.

The PEG and the activating agent used in the chemical modification withthe PEG and the activating agent, and the like may be selected fromthose commonly used in the art.

The site of modification (bonding) with the PEG in the peptidechemically modified with PEG of the present invention is notparticularly limited. The site of modification, however, is preferably asite other than the sites of the bonding of the peptide used in thepresent invention with the peptide-binding substance such as nucleicacid, and phosphatidyl serine or the like, and more preferably, the siteof modification is at N terminal, C terminal, or on the side chain.

The method used for introducing the PEG into the desired site in thepeptide may be any method known in the art.

The amount of PEG for the modification of (namely, PEG to be bonded to)the peptide used in the present invention is not limited to anyparticular amount.

By modifying the peptide used in the present invention with theactivated PEG, improvements of the characters and properties (which aredescribed below) inherent to the original peptide have been enabled.

Such improvements enabled for the peptide used in the present inventioninclude improvement in the rate of incorporation of the genes and thedrugs into the target cell, improvement in the pharmacological activity,decrease in the toxicity, and other excellent effects.

Next, “the amino acid sequence comprising 18 amino acids”, which is astructure critical in the peptide used in the present invention that isa peptide including a sequence of 18 amino acids, is described byreferring to the drawings.

Edmundson wheel plot is a model which shows position of the amino acidsin relation to the central axis of the α-helix, and this plot isdepicted so that the wheel is completed by 18 amino acids and the 19thamino acid comes to the same position as the 1st amino acid.

In the model depicted by this method, the first amino acid located atthe starting point is typically depicted at the position of 12 o'clockin a clock. There is, however, no difference in the relative location ofthe amino acids in the plot if the plotting were started from adifferent position in the drawing as long as the amino acid sequence isthe same. For example, (A), (B), and (C) are essentially identical inFIG. 1.

In the present invention, the “side” designates an area of the modelconstituted by consecutive and adjacent amino acids. The number of aminoacids constituting each side may be one or more, and preferably, eachside may comprise two or more amino acids.

“Consecutive and adjacent” designates, for example, the positionalrelation how the 1st and the 12th amino acids, or four amino acids,namely, the 15th, the 8th, the 1st, and the 12th amino acids are locatedin FIG. 1. In contrast, the 1st and the 10th amino acids are not locatedin consecutive manner. The 1st and the 5th amino acids are also notlocated in “consecutive and adjacent” manner, while the 1st and 5thamino acids may constitute the same side together with the adjoining12th amino acid.

The “hydrophobic side” is a side which includes a substantial number ofhydrophobic amino acids. The hydrophobic amino acid is not particularlylimited as long as it is substantially hydrophobic, and the hydrophobicamino acid may be a natural hydrophobic amino acid, or a modified orsynthetic amino acid having characteristic features nearly equivalent tothose of the natural amino acid.

The peptide used in the present invention may preferably include 80 mole% or more of hydrophobic amino acids in the hydrophobic side, and morepreferably, no acidic hydrophilic amino acid (aspartic acid and glutamicacid) in the hydrophobic side since the acidic hydrophilic amino acidinterferes with the electrostatic binding formed between the positivelycharged moiety of the peptide and the negatively charged moiety of thenucleic acid.

The hydrophobic amino acids are preferably those selected from leucine,isoleucine, valine, tryptophan, proline, tyrosine, alanine,phenylalanine, methionine, cysteine, and glycine. One side (side A) ofthe hydrophobic sides may preferably comprise 5 to 7 amino acids. Theother hydrophobic side (side C) may preferably comprise 2 to 4 aminoacids. It is particularly preferable that side C comprises solely fromhydrophobic amino acids.

Typical hydrophobic sides (side A and side C) are shown in FIG. 2.

The “hydrophilic side” is a side which includes a substantial number ofhydrophilic amino acids. The hydrophilic amino acid is not particularlylimited as long as it is substantially hydrophilic, and the hydrophilicamino acid may be a natural hydrophilic amino acid, or a modified orsynthetic amino acid having characteristic features nearly equivalent tothose of the natural amino acid.

The “positively charged side” is a “hydrophilic side” which includes aconsiderable number of substantially positively charged hydrophilicamino acids. The hydrophilic amino acid may be a substantiallypositively charged, natural hydrophilic amino acid, or a modified orsynthetic amino acid having characteristic features nearly equivalent tothose of the natural amino acid.

The peptide used in the present invention may preferably contain 80 mole% or more of hydrophilic amino acids in the hydrophilic side, and thehydrophilic amino acids are preferably those selected from asparagine,glutamine, threonine, serine, arginine, histidine, lysine, asparticacid, and glutamic acid. It is more preferable that the hydrophilicamino acids are those other than acidic hydrophilic amino acids, namely,those selected from asparagine, glutamine, threonine, serine, arginine,histidine, and lysine since the acidic hydrophilic amino acid interfereswith the electrostatic binding formed between the positively chargedmoiety of the peptide and the negatively charged moiety of the nucleicacid.

One side (side B) of the hydrophilic sides is preferably a positivelycharged side comprising 5 to 6 amino acids. More preferably, 50 mole %or more of the amino acids constituting this side are selected fromlysine and arginine.

The other hydrophilic side (side D) may preferably comprise 3 to 5 aminoacids.

Typical hydrophilic side (side D) and positively charged side (side B)are shown in FIG. 2.

It is to be noted that the “mole %” used herein designates the ratio ofthe number of hydrophobic amino acids in the hydrophobic side to thenumber of amino acids constituting the hydrophobic side, the ratio ofthe number of hydrophilic amino acids in the hydrophilic side to thenumber of amino acids constituting the hydrophilic side, or the ratio ofthe number of amino acids selected from lysine and arginine in thepositively charged side to the number of amino acids constituting thepositively charged side.

The four sided structure comprising the alternately arranged twohydrophobic sides and two hydrophilic sides (wherein at least one of thehydrophilic sides is a positively charged side) which is thecharacteristic feature of the peptide used in the present invention is astructure wherein, when the 18 amino acids shown in the model is dividedinto the sides in accordance with the definition as described above, twohydrophobic sides (side A and side C) and two hydrophilic sides (side Band side D, wherein at least side B is a positively charged side) arealternately arranged to constitute the four side.

It is to be noted that, when a hydrophobic side is directly juxtaposedto another hydrophobic side, these sides are not regarded as twohydrophobic sides but as one integrated hydrophobic side; and when ahydrophilic side is directly juxtaposed to another hydrophilic side,these sides are regarded not as two hydrophilic sides but one integralhydrophilic side; and when a positively charged side is directlyjuxtaposed to another positively charged side, these sides are regardednot as two positively charged sides but as one integral positivelycharged side.

In other words, in the four sided structure, the hydrophobic side is notadjacent to another hydrophobic side, the hydrophilic side is notadjacent to another hydrophilic side, and the positively charged side isnot adjacent to another positively charged side. “The structure whereintwo hydrophobic sides and two hydrophilic sides are alternately arrangedto constitute the four side” is not particularly limited as long as thefour sides are arranged [side A→side B=side C→side D (→side A)] inclockwise or [side A→side D→side C→side B (→side A)] in clockwise in theα-helix structural model of Edmundson wheel plot, and the function isretained. The preferable sequence is the one wherein sides are arranged[side A→side B→side C→side D (→side A)] in clockwise. It is to be notedthat the four sided structure is not limited for its method how theamino acids are divided, namely, how the 18 amino acids are allocated toeach of four sides as long as each side retains its character.

The amino acids are preferably allocated on the basis of the amino acidsequence such that side A comprises 5 to 7 amino acids, side B comprises5 to 6 amino acids, and side C comprises 2 to 4 amino acids, and side Dcomprises 3 to 5 amino acids.

Typical three sequences wherein the amino acid sequence is allocated toeach side in a different manner are shown as sequence A to C (C1 and C2)in FIGS. 3 and 4. It is to be noted that the amino acid sequence of C1and C2 is completely the same, and the different allocations are bothwithin the scope of the definition as described above. The allocation ofamino acids in A to C (C1 and C2) are as described below. HydrophobicHydrophilic Hydrophobic Hydrophilic side side side side (Side A) (SideB) (Side C) (Side D) A 5 6 2 5 B 6 5 4 3 C1 6 5 3 4 C2 7 5 3 3

Since the peptide used in the present invention contains the amino acidsequence comprising 18 amino acids exhibiting the four sided structureas its characteristic feature as described above, the peptide hasexcellent solubility in water. The peptide also has a characteristicfeature that it is capable of forming a complex with a peptide-bindingsubstance without forming aggregates of the complex which may cause asubstantial problem.

In contrast to the peptide vector having an α-helix structure which hasbeen described in the section of “Prior Art”, in the case of the peptideused in the present invention, a plurality of hydrophobic sides areformed when the peptide takes α-helix structure, and therefore, thepeptide has high ability of introducing a peptide-binding substance intothe cell even when the proportion of the hydrophobic sides is reducedfor the purpose of improving the solubility in water. A plurality ofhydrophilic sides are also formed simultaneously, and accordingly,proportion of the hydrophilic sides is higher compared to the peptide oftwo sided structure, and the hydrophilicity of the peptide does notcompletely disappear even when the peptide-binding substance becomeselectrostatically bonded to the at least one positively charged side ofthe peptide. Solubility in water of the complex of the peptide and thepeptide-binding substance is thereby maintained, and as a consequence,formation of troublesome aggregate masses is prevented. As describedabove, if the peptide were to have a high peptide-bindingsubstance-introducing ability simultaneously with an excellentsolubility in water, a plurality of hydrophobic sides and a plurality ofhydrophilic sides (of which at least one side is the positively chargedside) need to be formed when the peptide takes α-helix structure.

The peptide chemically modified with PEG of the present invention, asincluding the peptide of above characters used in the present invention,is further improved in such characters and properties.

It should also be noted that the peptide is not limited for its numberof sides as long as two or more hydrophobic sides and two or morehydrophilic sides are formed, and the function as a peptide vector ismaintained. However, the peptide may preferably have two hydrophobic andtwo hydrophilic sides, and in particular, at least one side of the twoor more hydrophilic sides is preferably a side rich in neutralhydrophilic amino acids (namely, a side with no substantial charge),since such side will not become bonded to the peptide-binding substanceand water solubility of such side will substantially be fullymaintained.

The peptide used in the present invention is by no means limited for itslength of the amino acid sequence as long as its function is retained.The peptide, however, is preferably the one having a total amino acidresidue number of 20 or more, more preferably 25 or more, and mostpreferably 30 or more. The peptide may preferably have a total aminoacid residue number of up to 100, more preferably up to 50, and mostpreferably up to 40.

The peptide used in the present invention includes at least one aminoacid sequence of 18 consecutive amino acids starting from any aminoacid. Preferably, the peptide of the present invention is a peptidecontaining at least two independent amino acid sequences of 18consecutive amino acids starting from any amino acid; and/or at leasttwo overlapping amino acid sequences of 18 consecutive amino acids. Morepreferably, the peptide of the present invention is a peptide whereinany consecutive 18 amino acids excluding the amino acids at the oppositeends represents the amino acid sequence of 18 amino acids exhibiting thefour sided structure of the present invention, that is, a peptidewherein all of the overlapping amino acid sequences comprising 18consecutive amino acids excluding the amino acids at the opposite endsexhibits the four sided structure of the present invention.

“The amino acid sequence comprising 18 amino acids” used hereindesignates an amino acid sequence comprising 18 amino acids wherein sideA, side B, side C, and side D are arranged in clockwise direction in theα-helix model by Edmundson wheel plot (hereinafter referred to as “thefour sided structure of the present invention”) (Such sequence ishereinafter referred to as “the amino acid sequence comprising 18 aminoacids exhibiting the four sided structure of the present invention”).

“The amino acid sequence comprising any consecutive 18 amino acidsexcluding the amino acids at opposite ends” means, for example, anyamino acid sequence in a peptide comprising N amino acids (wherein Nrepresents a number of 20 or more) comprising 2nd to 19th, 3rd to 20th,4th to 21st, or in a similar manner, (N-18)th to (N-1)th amino acids.“All of the overlapping amino acid sequences comprising 18 consecutiveamino acids excluding the amino acids at the opposite ends exhibits thefour sided structure of the present invention” means that all of theabove-mentioned sequences comprising 2nd to 19th, 3rd to 20th, 4th to21st, or in a similar manner, (N-18)th to (N-1)th amino acids exhibitthe four sided structure of the present invention. Examples of suchamino acid sequence are shown in FIGS. 5 and 6 as sequences A to D inthe α-helix model by Edmundson wheel plot, and the sequences shown arethe sequences comprising 18 amino acids of from 2nd to 19th, from 3rd to20th, from 4th to 21st, and from 19 to 36th amino acids in the peptideof SEQ ID NO: 16. All of these sequences show the four sided structureof the present invention.

In view of further improving the solubility of the complex of thepeptide and the substance which binds to the peptide, the peptide usedin the present invention is preferably the one wherein at least one endcomprises a hydrophilic amino acid, and more preferably, the one whereinboth ends comprise a hydrophilic amino acids.

Exemplary such peptide-binding substances include nucleic acids, acidichigh molecular weight compounds such as acidic protein, andphysiologically active low molecular weight compounds having anegatively charged side chain (as will be further described below).

The hydrophilic amino acid is not particularly limited as long as it ishydrophilic. The hydrophilic amino acid, however, is preferably the oneother than acidic hydrophilic amino acid, and more preferably, a neutralhydrophilic amino acid, and most preferably threonine or serine.

Preferable examples of “the amino acid sequence of 18 amino acidsexhibiting the four sided structure of the present invention” includedin the peptide used in the present invention are the amino acidsequences comprising any consecutive 18 amino acids in the followingamino acid sequence:

-   -   X2-X3-X4 -X5-X6-X7-X8-X9-X10-X11-X12-X13-X14        -X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-X32-X33-X34-X35-X36,        provided that,

in each of “X4, X8, X11, X15, and X19”, “X8, X11, X15, X19, and X22”,“X11, X15, X19, X22, and X26”, “X15, X19, X22, X26, and X29”, and “X19,X22, X26, X29, and X33”, at least 4 amino acids in the 5 amino acids area hydrophobic amino acid,

X3, X10, X12, X21, X28, and X30 are independently a member selected froma hydrophobic amino acid, a neutral hydrophilic amino acid and a basichydrophilic amino acid,

in each of “X2, X5, X9, X13, and X16”, “X5, X9, X13, X16, and X20”, “X9,X13, X16, X20, and X23”, “X13, X16, X20, X23, and X27”, “X16, X20, X23,X27, and X31”, and “X20, X23, X27, X31, and X34”, at least 4 amino acidsin the 5 amino acids are a neutral hydrophilic amino acid or a basichydrophilic amino acid, at least 3 amino acids of which being arginineor lysine,

X6, X17, X24, and X35 are independently a hydrophobic amino acid, and

X7, X14, X18, X25, X32, and X36 are independently a neutral hydrophilicamino acid or a basic hydrophilic amino acid.

Preferably,

X8 and X26 are proline,

X4, X17, X22, and X35 are leucine,

X6, X11, X15, X24, X29, and X33 are independently a hydrophobic aminoacid,

X12, X19, and X30 are independently a hydrophobic amino acid or aneutral hydrophilic amino acid,

X2, X5, X9, X20, X23, and X27 are independently a basic hydrophilicamino acid,

X13 and X31 are independently a basic hydrophilic amino acid or aneutral hydrophilic amino acid,

X16 and X34 are independently a hydrophobic amino acid or a basichydrophilic amino acid,

in each of “X2, X5, X9, X13, and X16”, “X5, X9, X13, X16, and X20”, “X9,X13, X16, X20, and X23”, “X13, X16, X20, X23, and X27”, “X16, X20, X23,X27, and X31”, and “X20, X23, X27, X31, and X34”, at least 3 amino acidin the 5 amino acids are arginine or lysine,

X3, X10, X21, and X28 are independently a member selected from ahydrophobic amino acid, a neutral hydrophilic amino acid, and a basichydrophilic amino acid, and

X7, X14, X18, X25, X32, and X36 are independently a neutral hydrophilicamino acid or a basic hydrophilic amino acid.

More preferably,

X2, X5, X9, X20, X23, and X27 are independently arginine or lysine,

X3 and X21 are independently a member selected from tyrosine,phenylalanine, serine, and arginine,

X4, X17, X22, and X35 are independently leucine,

X6, X15, X24, and X33 are independently leucine or isoleucine,

X7, X13, X25, and X31 are independently histidine or arginine,

X8 and X26 are independently proline,

X10 and X28 are independently a member selected from serine, arginine,and leucine,

X11 and X29 are independently tryptophan or leucine,

X12 and X30 are independently valine, leucine, or serine,

X14 and X32 are independently a member selected from glutamine,asparagine, and arginine,

X16 and X34 are independently alanine or arginine,

X18 is a member selected from arginine, lysine and serine,

X19 is leucine or threonine, and

X36 is arginine or serine.

In this connection, the sequence of amino acids X2 to X36 may includedeletion, addition, insertion, or substitution as long as at least 18amino acids are conserved in consecutive form.

It should be noted that, in the above description, a hydrophobic aminoacid is an amino acid selected from leucine, isoleucine, valine,tryptophan, proline, tyrosine, alanine, cysteine, phenylalanine,methionine, and glycine; a basic hydrophilic amino acid is an amino acidselected from arginine, histidine, and lysine; and a neutral hydrophilicamino acid is an amino acid selected from asparagine, glutamine,threonine, and serine.

A preferable example of the peptide used in the present invention is apeptide comprising the following amino acid sequence:

-   -   X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-X32-X33-X34-X35-X36-X37,        provided that

X1 and X37 are a hydrophilic amino acid,

in each of “X4, X8, X11, X15, and X19”, “X8, X11, X15, X19, and X22”,“X11, X15, X19, X22, and X26”, “X15, X19, X22, X26, and X29”, and “X19,X22, X26, X29, and X33”, at least 4 amino acids in the 5 amino acids area hydrophobic amino acid,

X3, X10, X12, X21, X28 and X30 are independently a member selected froma hydrophobic amino acid, a neutral hydrophilic amino acid, and basichydrophilic amino acid,

in each of “X2, X5, X9, X13, and X16”, “X5, X9, X13, X16, and X20”, “X9,X13, X16, X20, and X23”, “X13, X16, X20, X23, and X27”, “X16, X20, X23,X27, and X31”, and “X20, X23, X27, X31, and X34”, at least 4 amino acidsin the 5 amino acids are a neutral hydrophilic amino acid or a basichydrophilic amino acid, at least 3 amino acids of which being arginineor lysine,

X6, X17, X24, and X35 are independently a hydrophobic amino acid, and

X7, X14, X18, X25, X32, and X36 are independently a neutral hydrophilicamino acid or a basic hydrophilic amino acid.

It is to be noted that in the amino acid sequence as described above,the sequence of amino acids X2 to X36 may include deletion, addition,insertion, or substitution as long as at least 18 amino acids areconserved in consecutive form.

Preferably,

X1 and X37 are independently threonine or serine,

X8 and X26 are independently proline,

X4, X17, X22, and X35 are independently leucine,

X6, X11, X15, X24, X29, and X33 are independently a hydrophobic aminoacid,

X12, X19, and X30 are independently a hydrophobic amino acid or aneutral hydrophilic amino acid,

X2, X5, X9, X20, X23, and X27 are independently a basic hydrophilicamino acid,

X13 and X31 are independently a basic hydrophilic amino acid or aneutral hydrophilic amino acid,

X16 and X34 are independently a hydrophobic amino acid or a basichydrophilic amino acid,

in each of “X2, X5, X9, X13, and X16”, “X5, X9, X13, X16, and X20”, “X9,X13, X16, X20, and X23”, “X13, X16, X20, X23, and X27”, “X16, X20, X23,X27, and X31”, and “X20, X23, X27, X31, and X34”, at least 3 amino acidsin the 5 amino acids is arginine or lysine,

X3, X10, X21, and X28 are independently a member selected from ahydrophobic amino acid, a neutral hydrophilic amino acid, and a basichydrophilic amino acid, and

X7, X14, X18, X25, X32, and X36 are independently a neutral hydrophilicamino acid or a basic hydrophilic amino acid.

It is to be noted that the sequence of amino acids X2 to X36 may includedeletion, addition, insertion, or substitution as long as at least 18amino acids are conserved in consecutive form.

More preferably,

X1 is threonine,

X37 is serine,

X2, X5, X9, X20, X23, and X27 are independently arginine or lysine,

X3 and X21 are independently a member selected from tyrosine,phenylalanine, serine and arginine,

X4, X17, X22, and X35 are independently leucine,

X6, X15, X24, and X33 are independently leucine or isoleucine,

X7, X13, X25, and X31 are independently histidine or arginine,

X8 and X26 are independently proline,

X10 and X28 are independently a member selected from serine, arginine,and leucine,

X11 and X29 are independently tryptophan or leucine,

X12 and X30 are independently a member selected from valine, leucine andserine,

X14 and X32 are independently a member selected from glutamine,asparagine and arginine,

X16 and X34 are independently alanine or arginine,

X18 is a member selected from arginine, lysine and serine,

X19 is leucine or threonine, and

X36 is arginine or serine.

It is to be noted that in the amino acid sequence as described above,the sequence of amino acids X2 to X36 may include deletion, addition,insertion, or substitution as long as at least 18 amino acids areconserved in consecutive form.

In the above description, the hydrophobic amino acid, the basichydrophilic amino acid, and the neutral hydrophilic amino acid are thesame as those described before for the amino acid sequence comprisingamino acids X2 to X36.

It is to be noted that the amino acid sequences as mentioned above areonly some examples of the peptide used in the present invention, and thepeptide used in the present invention may be of any amino acid sequenceas mentioned above including deletion, addition, insertion, orsubstitution as required as long as the peptide includes “the sequenceof 18 amino acids exhibiting the four sided structure of the presentinvention” and retains the function of its own to thereby ensure thefunction of the peptide chemically modified with PEG.

If necessary, the peptide may also be modified with a molecule otherthan amino acid as long as the function of the peptide chemicallymodified with PEG is ensured. For example, the peptide may be modifiedwith a sugar chain, a lipid, or a high molecular weight compound inorder to increase in vivo stability, and/or by a sugar chain, a lipid,or a high molecular weight compound in order to suppress the recognitionof the peptide by an antigen presenting cell. To be more specific, thepeptide may be modified with mannose or cholesterol, and such modifiedpeptides are also comprehended in the peptide used in the presentinvention.

The peptide used in the present invention does not contain any acidichydrophilic amino acid, and this feature is particularly useful when thepeptide is to be modified. To be more specific, such peptide can bedesigned to include no acidic amino acid and to include carboxyl grouponly at its C terminal, and when the peptide is modified by utilizing areaction depending on the carboxyl group, a site specific modificationof the C terminal is enabled.

When the peptide used in the present invention is to besite-specifically modified by utilizing thiol group in cysteine residue,it is advantageous to design the peptide so that only one cysteine ispresent in the amino acid sequence, and preferably, so that the cysteineis added at the N terminal or C terminal of the peptide becauseselective modification of the peptide is enabled, for example, in themodification with polyethylene glycol according to the presentinvention.

By the way, amino acids are categorized by the type and nature of theirside chain molecules, and the categorization which may serve animportant index in elucidating higher order structure of the peptide isthe categorization based on the polarity of the side chain molecule. Tobe more specific, the amino acids are categorized as described below.

(1) Hydrophobic amino acid: glycine, alanine, valine, leucine,isoleucine, methionine, phenylalanine, tyrosine, cysteine, tryptophan,and proline

(2) Acidic hydrophilic amino acid: aspartic acid, and glutamic acid

(3) Neutral hydrophilic amino acid: serine, threonine, glutamine, andasparagine

(4) Basic hydrophilic amino acid: arginine, lysine, and histidine

Accordingly, in the peptide used in the present invention, amino acidswhich belong to the same category are mutually replaceable as long asthe requirement that “the amino acid sequence of 18 amino acids exhibitsthe four sided structure of the present invention” is fulfilled. Forexample, isoleucine and leucine, valine and leucine, tyrosine andphenylalanine, tryptophan and leucine, asparagine and glutamine, serineand threonine, arginine and lysine, and histidine and lysine aremutually replaceable.

In the case of histidine which is categorized as a member of basichydrophilic amino acids, it is only weakly charged under particularconditions, for example, under the physiological conditions, and itshares the nature similar to that of a neutral hydrophilic amino acid,and therefore, histidine is not only replaceable with a basichydrophilic amino acid, but also with a neutral hydrophilic amino acid.

In the meanwhile, the amino acids which belong to different categoriesare also mutually replaceable as long as the requirement that “the aminoacid sequence of 18 amino acids exhibits the four sided structure of thepresent invention” is fulfilled.

Typical examples of such replacements are as described below.

(1) Replacement between a hydrophobic amino acid (neutral) and a neutralhydrophilic amino acid (which is equivalent to replacement betweenarbitrary neutral amino acids)

Examples: leucine and threonine; leucine and serine; valine and serine;and tyrosine and serine.

(2) Replacement between a hydrophobic amino acid (neutral) and a basichydrophilic amino acid (which is a replacement between opposites,namely, hydrophobic/hydrophilic and neutral/basic amino acids, and whichis equivalent to the replacement between any amino acids other thanacidic hydrophilic amino acids)

Examples: alanine and arginine; and tyrosine and arginine.

(3) Replacement between a neutral hydrophilic amino acid and a basichydrophilic amino acid (which is equivalent to the replacement betweenany hydrophilic amino acids other than acidic hydrophilic amino acids)

Examples: serine and arginine; and glutamine and arginine.

Furthermore, two or more of the amino acid replacements as describedabove may be combined as long as the requirement that “the amino acidsequence of 18 amino acids exhibits the four sided structure of thepresent invention” is fulfilled. An exemplary combination of the aminoacid replacements between the amino acids of the same category is thereplacement of isoleucine with leucine combined with the replacement ofvaline with leucine. An exemplary combination of the amino acidreplacements between the amino acids of different categories is thereplacement of tyrosine with serine combined with the replacement ofserine with leucine. An exemplary combination of the amino acidreplacement between the amino acids of the same category and the aminoacid replacements between the amino acids of different categories is thereplacement of isoleucine with leucine combined with the replacement ofleucine with threonine. The number of the amino acid replacements thatmay be combined is not limited as long as the requirement that “theamino acid sequence of 18 amino acids exhibits the four sided structureof the present invention” is fulfilled. However, the number of aminoacid replacements combined is preferably 3 or less per 18 amino acids.

For example, Examples 12 and 13 demonstrate that the peptide used in thepresent invention can include amino acid replacements as long as therequirement that “the amino acid sequence of 18 amino acids exhibits thefour sided structure of the present invention” is fulfilled.

A typical peptide used in the present invention is a peptide having anyof the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 24, and thepeptide used in the present invention is preferably a peptide having theamino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 19.

The peptide used in the present invention is a peptide which has anability of binding to a nucleic acid and an ability of introducing thenucleic acid into a cell, and which also has a specific affinity forphosphatidyl serine.

The amino acids constituting the peptide may be either L- or D-aminoacids, and may be amino acids other than typical amino acids orsynthetic, modified amino acids as long as they substantially sharecommon nature with the natural amino acids. Exemplary such amino acidsinclude hydroxyproline, homoserine, and methylcysteine.

The present invention provides the peptide chemically modified with PEGthat is obtained by modifying the peptide including the amino acidsequence comprising 18 amino acids as described above (the peptide usedin the present invention) with PEG, and a complex formed of the peptidechemically modified with PEG and the substance which binds to thepeptide (the peptide-binding substance) is also within the scope of thepresent invention.

The second aspect of the present invention is the complex formed of thepeptide chemically modified with PEG and the substance which binds tothe peptide (the peptide-binding substance).

Such “complex” may be an aggregate, mixture or composition comprisingthe peptide used in the present invention, PEG, and the peptide-bindingsubstance.

The site in the peptide used in the present invention to which PEGand/or the peptide-binding substance is to be bonded is not particularlylimited as long as the function of the peptide is not impaired.

The results of Example 30 reveal that a complex (such as aggregate,mixture, composition, or the like) is formed between the peptide used inthe present invention, PEG, and the peptide-binding substance.

As described above, the “peptide-binding substance” constituting thecomplex of the present invention may be a nucleic acid, an acidic highmolecular weight compound such as acidic protein, or a physiologicallyactive low molecular weight compound having a negatively charged sidechain.

Examples of “acidic high molecular weight compounds such as acidicprotein” include proteins which are rich in acidic amino acids and whichare negatively charged (for example, albumin), and high molecular weightcompounds other than proteins wherein the entire molecule is negativelycharged (for example, heparin and hyaluronic acid).

Examples of “low molecular weight compounds having a negatively chargedside chain” include a low molecular weight compound having phosphategroup or the like on its side chain such as phosphorylated acyclovir.

The “nucleic acid” includes a nucleoside, a nucleotide, anoligonucleotide or a polynucleotide comprising two or more nucleotides,a DNA, an RNA, a derivative thereof, a modification thereof, and ananalog thereof.

The “nucleic acid derivative” includes a nucleic acid wherein some ofthe atoms constituting the nucleic acid have been replaced with otheratoms. An example of such “nucleic acid derivative” is the PS formwherein one of the oxygen atoms in the-phosphodiester bond moiety hasbeen replaced with sulfur atom.

The “modified nucleic acid” includes a nucleic acid wherein some of theatoms constituting the nucleic acid have been replaced with other atomicgroup or some of the atoms constituting the nucleic acid have otheratomic group added thereto. Examples of such “modified nucleic acid” arethe one wherein the carbon atom located at 2′ position of the pentosemoiety in the nucleic acid has methoxy group (—O—CH₃) added thereto, andthose wherein the nucleic acid sequence has a sugar, a phospholipid, orpolyethylene glycol added thereto in some part thereof.

The “nucleic acid analog” includes a molecule which has a backboneentirely different from that of a nucleic acid while the moleculeretains the function expected from the nucleic acid. An example of such“nucleic acid analog” is a peptide nucleic acid (PNA). In this respect,the nucleic acid also comprehends therein a DNA or an RNA which is apolynucleotide that increases or decreases the amount of particularprotein expressed in the body, or regulates the expression of thefunction of particular factor in the body; a derivative, a modification,or an analog of such DNA or RNA; a combination of such derivative,modification and analog; and a mixture or chimera of such derivative,modification, or analog.

Furthermore, the nucleic acid as described above may be a singlestranded nucleic acid or a nucleic acid of two or more strands, and maybe the one bound to a carrier. For example, the nucleic acid may be theDNA coding for a protein, a plasmid wherein an expression-regulatingunit has been linked to such DNA, an antisense oligonucleotide, adouble-stranded nucleic acid serving as a decoy (hereinafter referred toas decoy), an aptamer, a ribozyme, or an siRNA.

The “particular protein” or the “particular factor” used hereindesignates a protein or a factor whose amount expressed is to beincreased or decreased, or whose expression is to be regulated by thenucleic acid. Such protein and factor may be either the one found in aliving body or the one not found in a living body.

The “ability of binding to a peptide-binding substance” can be assayed,when explained by using a nucleic acid as an example, by subjecting amixture of the nucleic acid and the peptide used in the presentinvention to electrophoresis, and detecting the image of the stainednucleic acid. For example, when the nucleic acid is not electrophoresedand the stained image of the nucleic acid is not detected, or when thestained image detected is in the region where distance of the migrationof the stained image is small compared to the stained image of thenucleic acid alone, the peptide can be determined to have “the abilityof binding to a peptide-binding substance”. Illustrative procedure ofsuch assay will be described in Example 8.

The “ability of introducing a peptide-binding substance into a cell” canbe measured, when explained by using a nucleic acid as an example, byobserving the cell under a fluorescence microscope using afluorescent-labeled nucleic acid, or by using a plasmid which expressesa reporter gene and measuring the reporter protein expressed by thecell. The ability can also be measured by using the pharmacologicalaction resulting from the expression of the reporter protein as theindex. Typical reporters include firefly luciferase, β-galactosidase,and HSV-tk, and illustrative procedure will be described in Example 9.When the amount of the firefly luciferase expressed by the cell ismeasured by such procedure, fluorescent count per 1 mg of protein per 1second is measured. When the fluorescent count is 10,000 or more, thereporter gene is determined to have been introduced into the cell, andthe peptide is determined to have the “ability of introducing apeptide-binding substance into a cell”.

It is to be noted that, in the present invention, the “introduction in(in/into/to) the cell” designates the same situation as the“introduction into the interior of the cell”.

In the present invention, “the interior of the cell” designates theunits constituting the cell and their interior. For example, included in“the interior of the cell” are the inside of the phospholipid bilayerconstituting the contour of the cell, the space between the two layersof the phospholipid bilayer, as well as cytoplasm, organella, nucleus,and their interior.

“Specific affinity” means that a peptide exhibits some specificinteraction or other, for example, binding, formation of complex, mutualrecognition of the molecule, tendency of moving in a particulardirection or being collected in a particular direction, change ofmolecular configuration, or mutual reaction. For example, a peptide isdetermined to have a specific affinity if the peptide exhibits affinityin the presence of serum albumin.

Even if a peptide interacts with a particular substance, the interactionis generally nonspecific if the interaction disappears in the presenceof other peptides or proteins. To be more specific, a nonspecificinteraction disappears in the presence of a large amount of albumin orother protein. Accordingly, a peptide which exhibits affinity forphosphatidyl serine in the absence of serum albumin but fails to exhibitaffinity for phosphatidyl serine in the presence of serum albumin isnonspecific with regard to the interaction with phosphatidyl serine, andthe peptide will exhibit no affinity for phosphatidyl serine in a livingbody.

On the other hand, when the interaction of a peptide with the particularsubstance does not disappear in the presence of other peptides orproteins, the interaction can be deemed specific, and the peptide can beregarded to have a “specific affinity”.

A “carrier” is a substance which binds to or incorporates a drug or thelike for its delivery. While the carrier is not limited to anyparticular type as long as it has such function, the carrier ispreferably the one comprising a lipid or a high molecular weightcompound, and the preferable examples include liposomes, dendrimers, andnanoparticles. Combination of the carrier and the drug is also notlimited, while the combination should be the one capable of holding thedrug in a stable manner. In this point of view, an electrostaticallyrepelling combination should preferably be avoided, and an exemplarypreferable combination is use of positively charged doxorubicin with aneutral or negatively charged liposome.

Use of the carrier modified with the peptide chemically modified withPEG of the present invention is particularly favorable since it hastargetability to a specific site in addition to an improved in vivokinetics. With regard to the peptide chemically modified with PEG, theactivated PEG used in the chemical PEG modification is not limited toany particular type as long as formation of the complex with the carrieris enabled. However, when the carrier contains a phospholipid as itsconstituent, the activated PEG is preferably the one having aphospholipid bonded thereto. The ratio of the number of molecules of thePEG not modified by the peptide to the number of molecules of thepeptide chemically modified with PEG of the present invention is notparticularly limited as long as in vivo kinetics of the carrier modifiedwith the peptide chemically modified with PEG is improved. However, whenthe carrier is a liposome, the ratio is preferably 1:1 to 1:0.001, morepreferably 1:0.3 to 1:0.01, and most preferably 1:0.2 to 1:0.02.

Next, the properties and characters of the peptide used in the presentinvention, as well as the improved properties and characters of thepeptide chemically modified with PEG of the present invention aredescribed by referring to the Examples.

It is to be noted that the improved properties and characters of thepeptide chemically modified with PEG of the present invention includethe properties and characters of the complex of the peptide chemicallymodified with PEG of the present invention and the peptide-bindingsubstance and the properties and characters of the carrier which hasbeen modified with the peptide chemically modified with PEG of thepresent invention.

The peptide used in the present invention (namely, the peptide includingthe amino acid sequence comprising 18 amino acids that is not modifiedwith PEG) and the peptide chemically modified with PEG of the presentinvention (both two peptides alike being hereafter referred to as “thepeptide of the present invention”) have the ability of binding to anucleic acid. Preferably, the peptides are each the one provided withthe ability of forming a complex with the nucleic acid by substantialintegration between the peptide and the nucleic acid without completelycompromising any of other abilities of the peptide such as the abilityof introducing the nucleic acid into a cell or the affinity forphosphatidyl serine in the presence of serum albumin, and the functionsinherent to the nucleic acid as well.

The peptide of the present invention is not limited for its mode ofbinding to the nucleic acid, and the binding may be accomplished, forexample, by electrostatic bonding, hydrophobic bonding, or covalentbonding.

The peptide of the present invention has ability of forming the complexas above to introduce thereby the nucleic acid that has become bonded tothe peptide into the interior of a cell. Preferably, the peptide of thepresent invention has ability of introducing the nucleic acid into acell without causing decomposition of the nucleic acid and with thedesired function of the nucleic acid maintained.

In the nucleic acid-introducing ability measurement of Example 9, thenucleic acid-introducing ability of the peptide used in the presentinvention, as being determined as the fluorescent count per 1 mg ofprotein per 1 second, has a specific measured value of at least 10,000.Preferably, the peptide used in the present invention is a peptide whichexhibits a nucleic acid-introducing ability of at least 100,000, morepreferably of at least 1,000,000.

Since the peptide of the present invention is provided with the featuresas described above, the nucleic acid introduced by using the peptide iscapable of exerting its desired functions in the cell once it isintroduced in the cell. For example, the exogenous gene inserted in theplasmid may become expressed in the cell to produce the desired protein,or the antisense oligonucleotide, decoy, aptamer, ribozyme, or the likemay suppress production of a particular physiologically activesubstance. It is to be noted that “the desired protein” not onlyincludes the final active form of the protein but also the precursor forsuch final form of the protein.

Examples 11 and 24 will illustrate embodiments wherein fireflyluciferase gene that has been inserted in a plasmid is introduced in acell by the peptide used in the present invention, and after itstranscription and translation, firefly luciferase is produced andaccumulated in the cell. Examples 16 and 25 will illustrate embodimentswherein thymidine kinase (hereinafter abbreviated as HSV-tk) gene fromherpes simplex virus that has been inserted in the plasmid is introducedin a tumor cell by the peptide used in the present invention, and afterits transcription and translation, HSV-tk is produced and accumulated inthe tumor cell to thereby enhance ganciclovir sensitivity of the tumorcell and exert pharmacological action (anti-tumor action).

Furthermore, a preferred embodiment of the peptide of the presentinvention is a peptide which exhibits specific affinity for phosphatidylserine in the presence serum albumin, and which does not exhibits anyaffinity for phospholipids other than phosphatidyl serine, for example,phosphatidyl choline.

The interaction between the preferred embodiment of the peptide of thepresent invention which has no affinity for a phospholipid other thanphosphatidyl serine and the phosphatidyl serine, namely, the bindingbetween the peptide of the present invention and the phosphatidyl serineis not limited to the binding through charge or the binding throughnon-specific binding, and the binding may also be the binding enabled bythe recognition of the molecular structure of the phosphatidyl serine bythe peptide.

This is demonstrated, for example, in Example 20 and Example 21 withrespect to the peptide used in the present invention.

As described above, phosphatidyl serine is the phospholipid which isincluded as a component constituting the lipid bilayer forming thesurface layer of a cell, and the proportion of the phosphatidyl serinefound in the outer layer and the inner layer of the lipid bilayer variesdepending on the condition of the cell. To be more specific,phosphatidyl serine is a phospholipid which is believed to increase itsproportion in the outer layer of the lipid bilayer in an abnormal cellsuch as a cell in the inflammatory lesion wherein the cell has beeninjured, denatured or activated. Therefore, the peptide of the presentinvention selectively binds to the abnormal cell, for example, the cellin the inflammatory lesion. Phosphatidyl serine is also a phospholipidwhich provides a “field” in a living body for the blood coagulationreaction to take place, or a mark when macrophage recognizes and eats anapoptotic cell. Therefore, the peptide of the present inventionselectively binds to the abnormal cell, for example, to the “field” in aliving body where blood coagulation reaction is in progress.

Thus, the peptide of the present invention is characterized by itsability of binding to a nucleic acid, its ability of introducing thenucleic acid to a cell, and its affinity for phosphatidyl serine in thepresence of serum albumin.

The peptide of the present invention is preferably a peptide which takesan irregular structure in an aqueous solution containing no solute oronly an inorganic salt but which takes the α-helix structure in thepresence of a particular substance.

The “particular substance” used herein designates a substance whichinteracts with the peptide of the present invention to promote thepeptide to take the α-helix structure, and an exemplary such substanceis an amphipathic substance, for example, a surfactant such as sodiumdodecyl sulfate (SDS) or a particular phospholipid such as phosphatidylserine.

α-helix structure in the higher order structure of a peptide can begenerally confirmed by measuring CD spectrum. To be more specific, whenα-helix structure is present in the higher order structure of a peptide,mean residue ellipticity in the CD spectroscopy takes the form of “W”which is characteristic to the α-helix structure wherein local minimumis found in two wavelength regions, namely, in the region at thewavelength of 205 to 210 nm and the region at the wavelength of 220 to225 nm. (See “Optical rotation of proteins” (Experimental methods inbiological chemistry 6), Hamaguchi, H. et al., Japan ScientificSocieties Press, 1979). It is to be noted that the proportion of theα-helix structure in the higher order structure of the peptide can becalculated by a predetermined calculation method from the mean residueellipticity that had been measured. Typical examples of such calculationinclude the method of Chen et al. (Y. H. Chen et al., Biochemistry Vol.11, 4120 (1972)), the method of Yang et al. (J. T. Yang et al., Anal.Chem., Vol. 91, 13 (1978)), and the method of Woody et al. (R. W. Woodyet al., J. Mol. Biol., Vol. 242, 497 (1994)).

The value calculated, however, varies by the method used for thecalculation, and therefore, it is recommended that the method employedfor the calculation is indicated together with the value calculated.

As described above, the preferred embodiment of the peptide of thepresent invention takes α-helix structure in the presence of aparticular substance. For example, such preferred peptide takes α-helixstructure through interaction with an amphipathic substance such as asurfactant or a particular phospholipid on the cell membrane such asphosphatidyl serine. This invites increase in the permeability of thepeptide or the substance containing the peptide through cell membrane,and smooth migration of the peptide or the substance containing thepeptide into the cell.

To be more specific, the preferred embodiment of the peptide of thepresent invention does not exhibit α-helix structure in the CDspectroscopy in an aqueous solution containing no solute or in anaqueous solution containing only an inorganic salt at a pH of 5 to 8,but which shows two local minimums in the mean residue ellipticity inthe CD spectroscopy in an aqueous solution containing 5 mM SDS at a pHof 5 to 8 at two wavelength regions, namely, at the wavelength region of205 to 210 nm and at the wavelength region of 220 to 225 nm, indicatingthe α-helix structure.

The peptide is preferably the one wherein the proportion of the α-helixstructure in the higher order structure is 25% or more when calculatedby the method of Chen et al. More preferably, the peptide is the onewherein the proportion of the α-helix structure is 30% or more, andstill more preferably, the one wherein the proportion of the α-helixstructure is 35% or more.

Such peptide of the present invention as above is found to take anα-helix structure in an aqueous solution in the presence of anamphipathic substance. In addition, since such peptide has affinity forphosphatidyl serine, such peptide selectively accumulates on the surfaceof an abnormal cell, for example, on the surface of an injured,denatured, or activated cell, and takes the α-helix structure throughinteraction with a phosphatidyl serine on the cell membrane, and thepeptide is then selectively incorporated in the cell.

The peptide of the present invention is preferably the one which doesnot form aggregates in the presence of a protein.

For example, in the case of the preferred embodiment of the peptide ofthe present invention, the peptide will not take an α-helix structure inthe absence of an amphipathic substance, and solubility will bemaintained in the presence of proteins. As a consequence, the peptide isprevented from forming aggregates, which may cause substantial problem,even when administered to a human, and the risk of the blood vesselocclusion is reduced with an increased safety. Example 26, for example,demonstrates the high safety of the peptide used in the presentinvention.

It should be noted that the peptide chemically modified with PEG of thepresent invention is more improved in safety.

Whether or not the peptide of the present invention forms the aggregatesto a substantial level in the presence of a protein can be determined byoptically measuring turbidity of an aqueous solution of serum albumincontaining the peptide, for example, by measuring absorption of theaqueous solution of serum albumin containing the peptide at a wavelengthof 340 nm to 660 nm, and in particular, at a wavelength of 600 nm.

The peptide of the present invention is useful as a peptide vectorbecause of its characteristic features that the peptide easily binds tothe nucleic acid, that it exhibits high solubility after forming thecomplex with the nucleic acid, that it is highly capable of introducingthe nucleic acid in the cell, that it demonstrates the high safety, andthat it has affinity for phosphatidyl serine in the presence of serumalbumin enabling its selective incorporation into the abnormal cell.

The “peptide vector” is a peptide which is capable of introducing thedesired substance into a cell, its derivative, its modification, or itsanalog.

The peptide of the present invention also has a characteristic featurethat it can prevent decomposition of the nucleic acid by a nuclease.Accordingly, a natural type oligonucleotide or polynucleotide (P═O form)which is easily decomposed by the nuclease in the blood or cell becomesresistant to the decomposition by the nuclease once the peptide of thepresent invention is bonded to such oligonucleotide or polynucleotide.

Whether or not the peptide imparts the nuclease resistance to thenucleic acid can be determined by allowing the complex of the nucleicacid and the peptide of the present invention to react with the nucleasein a solution containing the nuclease, extracting the nucleic acid, andsubjecting the extracted nucleic acid to electrophoresis and detectingthe stained image. For example, if the peptide imparts the nucleic acidwith the nuclease resistance, the nucleic acid will remain intact andthe stained image of the nucleic acid will be obtained. Examples 18 and19 recite the detailed procedure.

When the peptide of the present invention is used as a peptide vector, asubstance capable of binding to the vector, which is preferably anucleic acid, can be introduced into the cell, and development of thefunction of the nucleic acid in the cell is realized by suchintroduction in the cell of the nucleic acid.

Examples 11 to 16 are directed to the embodiments wherein the peptide ofthe present invention is used to an in vitro introduction of a nucleicacid into the cell, and the firefly luciferase and HSV-tk coded by thenucleic acid are respectively expressed in the cell. Examples 24 and 25are directed to the embodiments wherein the peptide used in the presentinvention is adapted to an in vivo introduction of a nucleic acid in thecell, and the firefly luciferase and HSV-tk coded by the nucleic acidare respectively expressed in the cell. In particular, Example 25 isdirected to the embodiment wherein the HSV-tk gene that has beenintroduced in the tumor cell by the peptide used in the presentinvention is expressed in the tumor cell, and ganciclovir sensitivity ofthe tumor cell is thereby enhanced to exhibit the pharmacological action(anti-tumor action). The dose of the plasmid used in this Example was 10μg, and this dose was by far smaller than the dose (150 μg) used insimilar pharmacological experiment using the conventional liposomevector (Aoki, K. et al., Hum. Gene Ther., Vol. 8, 1105 (1997)). As willbe more illustratively shown in Examples 17 and 18, the peptide used inthe present invention increases the stability of the nucleic acid to anextent further than the liposome, and the peptide used in the presentinvention that has become bonded to the nucleic acid has a stabilityhigher than that of the liposome, and therefore, use of the peptide usedin the present invention in introducing a nucleic acid in a cell isuseful, and the peptide used in the present invention is excellent foruse as a vector in introducing a nucleic acid in a cell.

The peptide chemically modified with PEG of the present invention thatincludes the peptide of excellent characters as above is more excellentin such characters.

Other exemplary uses of the peptide of the present invention as a vectorinclude use of the peptide in introducing the following substances intothe cell to thereby allow development in the cell of the function of thefollowing substances:

a plasmid expressing a reporter protein (green fluorescent protein(GFP), β-galactosidase, etc.);

a plasmid expressing a cytokine (interleukin 2, interferon β, etc.)which exhibits anti-tumor effects;

a plasmid expressing a physiologically active substance (Fas ligand,p53, caspase 3, caspase 8, Bax (Bcl-2-associated X protein), FADD (Fasassociated death domain protein), etc.) which induces apoptosis to exertcytotoxic effects;

a plasmid expressing a soluble receptor for a ligand such as TNF-α orinterleukin 6, which competitively binds to the ligand to suppress thereaction induced by the ligand, and which can thereby improve thesymptom of, for example, chronic articular rheumatism;

a plasmid expressing a peptide/polypeptide which can serve a vaccine tosuppress an allergic reaction or a protein such as mite antigen which isan antigenic protein;

a plasmid expressing vascular endothelial growth factor (VEGF) orhepatocyte growth factor (HGF) which has the action of improving thepathological condition of arteriosclerosis obliterans as a circulatorydisease or promoting the healing (remodeling) of the injured lesion;

an antisense oligonucleotide or a ribozyme for CDC2 kinase which has theaction of suppressing the restenosis after percutaneous transluminalcoronary angioplasty (PTCA); and

a decoy for a nucleotide sequence of to which E2F (a transcriptionalregulatory factor for a cell cycle regulatory gene) or NFκB (atranscriptional regulatory factor for a cytokine) binds; as well as

a phosphorylated nucleic acid analog in its active form which exhibitsan anti-virus action, and the like.

Since the peptide of the present invention has the ability of binding toa nucleic acid, ability of introducing the nucleic acid to a cell, andaffinity for phosphatidyl serine in the presence of serum albumin as itscharacteristic features, the peptide will introduce the nucleic acid ata higher efficiency to a cell wherein a larger amount of phosphatidylserine has been translocated to the surface. This means that, when anucleic acid such as a gene or an antisense DNA is to be introduced in acell for the purpose of treating a disease, the nucleic acid will beselectively introduced in a larger amount to an abnormal cell such as aninjured, denatured, or activated inflammatory cell, or to animmunocompetent cell wherein an increased amount of phosphatidyl serinehas been translocated to the cell surface. Reduction of the side effectsis thereby attained.

For example, when the peptide of the present invention is used as avector in introducing a nucleic acid to a tumor cell for the purpose oftreating a cancer, the nucleic acid is scarcely introduced to a normalcell, while the nucleic acid is introduced to the tumor cell at a highrate. When a nucleic acid is introduced in a cell for the purpose oftreating an allergy, the nucleic acid is specifically introduced to acell wherein allergic reaction has been induced by the immunocompetentcell.

Furthermore, the nucleic acid can be introduced at a higher rate to thedesired particular cell or organ by utilizing the capability of thepeptide of the present invention “to introduce the nucleic acid at ahigher rate to the cell wherein a larger amount of phosphatidyl serinehas been translocated to the cell surface”, and namely, by preliminarilyincreasing the amount of the phosphatidyl serine translocated to thesurface of the desired particular cell or organ. To be more specific,introduction of the nucleic acid at a higher rate to the desiredparticular cell or organ can be realized by reacting a reagent with theparticular cell or organ to thereby increase the amount of thephosphatidyl serine translocated to the surface of the particular cellor organ by the pharmacological action of the reagent, and thereafterusing the peptide of the present invention as a vector.

For example, a therapy is still possible even if a chemotherapeutictreatment of a tumor by sole administration of an anticancer drug failedto achieve sufficient therapeutic effects, and in such a case, thechemotherapeutic agent may be administered to increase the amount of thephosphatidyl serine translocated to the surface of the tumor cell by thepharmacological action of the chemotherapeutic agent, and then, thepeptide of the present invention may be used as a vector to therebyrealize the highly efficient introduction of a gene, the antisense DNA,or other nucleic acid which exhibits high anti-tumor effects into thetumor cell and utilize the improved therapeutic effects of the nucleicacid.

As described above, the peptide of the present invention readily bindsto a nucleic acid and introduces the nucleic acid into the cell. Thepeptide of the present invention, however, is not only capable ofintroducing a nucleic acid but also capable of introducing anotherpeptide-binding substance into a cell. As in the case of the binding ofthe peptide of the present invention with the nucleic acid wherein themode of the binding is not particularly limited, the mode of the bindingis not limited in the case of the binding of the peptide of the presentinvention with such substance. The mode of the binding between thepeptide of the present invention and such substance upon introduction ofsuch substance into the cell is preferably a noncovalent bond, morepreferably an electrostatic bond, and most preferably an electrostaticbond established between the positive charge of the peptide of thepresent invention and the negative charge of the substance. In otherwords, the present invention includes within its scope an introductionof a peptide-binding substance into a cell based on the mode of thebinding as described above.

It is to be noted that, while the properties and characters of thepeptide used in the present invention and the peptide chemicallymodified with PEG of the present invention have been described together,the peptide chemically modified with PEG of the present invention is thepeptide used in the present invention which has been chemically modifiedwith the PEG, and it exhibits improved efficiency in incorporating thegene or the drug into the target cell, improved pharmacologicalactivity, reduced toxicity, and other favorable effects compared to thepeptide used in the present invention.

More specifically, as evident from the results of Examples 28 and 29 aswill be described below, specific activity is not impaired when thepeptide used in the present invention is modified with the activated PEG(Example 28), and the introduction rate of the peptide-binding substanceincreases about three times (Example 29). The usefulness of the presentinvention is thereby demonstrated.

As evident from the results of Examples 35 to 37 as will be describedbelow, the carrier which is modified with the peptide chemicallymodified with PEG of the present invention is capable of improving theintroduction rate of the drug incorporated in the carrier by 3 to 33folds (Examples 35 and 36), and extending the survival period of thecancer bearing mouse (Example 37). The usefulness of the presentinvention is thereby demonstrated.

In the following, it is described how the peptide chemically modifiedwith PEG of the present invention is produced.

The peptide used in the present invention can be produced by chemicalsynthesis.

For example, the peptide is obtained by a synthesis using an automaticpeptide synthesizer (432A, manufactured by Applied Biosystems).

The peptide used in the present invention may also be produced by agenetic engineering means. When the peptide is produced by geneticengineering methods, the desired peptide may be produced by thefollowing steps:

(1) the step of producing a DNA having the nucleotide sequence codingfor the amino acid sequence of the peptide;

(2) the step of introducing the DNA in a vector to thereby produce anamplifiable recombinant DNA including the DNA;

(3) the step of transforming a host cell with the recombinant DNA toproduce a transformant capable of expressing the peptide; and

(4) the step of cultivating the transformant to produce the peptide, andrecovering the peptide from the culture mixture.

The DNA coding for the peptide used in the present invention may be anyDNA having the nucleotide sequence which substantially codes for thepeptide used in the present invention. As is well known in the art,because of the degeneration of codon, at least one nucleotide in thegene sequence can be replaced with another nucleotide without causingany change in the amino acid sequence of the peptide coded by the genesequence. Therefore, the DNA may have a nucleotide sequence wherein atleast one nucleotide in the nucleotide sequence has been replaced on thebases of the degeneration of the genetic code. To be more specific, whenthe peptide used in the present invention is produced by using geneticengineering methods, the peptide may have a nucleotide sequence whereinat least one nucleotide has been replaced so that the codon will be theone frequently found in a particular host cell. In addition, the DNA maybe a recombinant DNA, such as a plasmid or an expression vector.

Exemplary DNAs of SEQ ID NO: 28 to SEQ ID NO: 30 coding for the peptidesof SEQ ID NO: 1, SEQ ID NO: 16, and SEQ ID NO: 19 are shown.

The step of obtaining the DNA having a nucleotide sequence coding forthe amino acid sequence of the peptide may be accomplished by thesynthesis using an automatic nucleic acid synthesizer.

The step of incorporating the DNA in the vector to obtain an amplifiablerecombinant DNA including the DNA, and the step of transforming the hostcell by the recombinant DNA to obtain a transformant which is capable ofexpressing the peptide may be accomplished by the genetic engineeringmethods generally used in the art as described in a book (for example,Molecular Cloning: a laboratory manual, Second edition, T. Maniatis etal., Cold Spring Harbor Laboratory Press (1989)). It is to be notedthat, since the peptide used in the present invention can be designed asa peptide including no methionine, the peptide can be obtained byproducing a peptide wherein a plurality of the peptides of the presentinvention are ligated by the intervening methionine by geneticengineering methods, and cleaving the peptide thus produced withcyanogen bromide.

The peptide produced may be purified, isolated, and recovered byreferring to methods described in various articles and books (forexample, “Experiments in Biochemistry: A New Lecture Series: Protein I”(the Japanese Biochemical Society, ed., Tokyo Kagaku Dozin, 1990),“Kagaku, Special edition, 102: high performance liquid chromatography ofproteins and peptides” (N. Ui et al., ed., Kagaku-Dojin, 1985)). To bemore specific, the peptide produced may be obtained in its pure form byusing at least one of the procedures selected from demineralization,concentration, salting out, ultrafiltration, ion exchangechromatography, reversed phase chromatography, isoelectricchromatography, affinity chromatography, and gel permeation.

The peptide chemically modified with PEG of the present invention isproduced by chemically modifying the peptide used in the presentinvention produced by the procedure as described above with the PEG asdescribed above.

The conditions of the chemical modification with the PEG is notparticularly limited, any adequate process may be selected from thoseknown to those skilled in the art.

Typically, the solution of the peptide in an adequate solvent and thesolution of the activated PEG in an adequate solvent are mixed, and themixture is allowed to react at about 1 to about 40° C. for about 1minute to about 24 hours.

The peptide chemically modified with PEG may also be purified after thechemical modification with the PEG by using the process and conditionscommonly used in the art.

In the production process as described above, the activated PEG is usedat an amount in molar ratio to the amount of the peptide used in thepresent invention of 0.0001 to 10000, and preferably 0.01 to 100, andmost preferably 0.1 to 10. When the activated PEG is used at an amountin such a range, the peptide chemically modified with PEG will retainthe activity of the original peptide while acquiring increasedsolubility, reduced antigenicity, reduced toxicity, and other favorablecharacters after the reaction of the activated PEG with the peptide.

The PEG moiety in the peptide chemically modified with PEG may have anaverage molecular weight of about 200 Da to about 100,000 Da, preferablyabout 1,000 Da to about 50,000 Da, and more preferably about 2,000 Da toabout 20,000 Da.

When the average molecular weight of the PEG moiety is within suchrange, the peptide modified with PEG will retain the activity of theoriginal peptide while acquiring increased solubility, reducedantigenicity, reduced toxicity, and other favorable characters.

Next, the process of producing the complex of the peptide chemicallymodified with PEG of the present invention and the peptide-bindingsubstance is described.

The complex is produced by using the peptide used in the presentinvention, the activated PEG, and the peptide-binding substance asdescribed above, by the process comprising the steps of:

I)a) reacting the peptide containing the sequence comprising 18 aminoacids with the activated polyethylene glycol (PEG), and

b) reacting the peptide chemically modified with PEG obtained in theabove step a) with the substance which binds to the peptide; or, by theprocess comprising the steps of:

II)a) reacting the peptide containing the sequence comprising 18 aminoacids with the peptide-binding substance, and

b) reacting the reaction product of the peptide and the peptide-bindingsubstance with the activated PEG.

The method II) is preferable since unnecessary PEG modification of thepeptide can be avoided by preliminarily forming the reaction product ofthe peptide with the peptide-binding substance, and modifying thereaction product with the PEG.

By employing the method II), unnecessary PEG modification such ascoverage in the PEG modification of the site required for the peptidefunctioning (coverage of the active center of the peptide), peptidestructure change that may hamper the approach of the peptide to thetarget, and other incidents that may lead to the reduced specificactivity can be avoided to further improve the usefulness of the PEGmodification.

The conditions used for the production of the peptide chemicallymodified with PEG of the present invention may be used for the reactionconditions of the step a) of method I), and the reaction conditionssimilar to those of method II) a) as will be described below may be usedfor the reaction conditions of step b) of method I).

The reaction conditions used in step a) of method II) is notparticularly limited, and any conditions enabling the formation of thereaction product may be selected. A typical condition is the conditionwherein the peptide and the peptide-binding substance that have beenrespectively dissolved in appropriate solvents are mixed at about 1 toabout 40° C. for about 1 minute to about 24 hours.

The reaction conditions used in step b) of method II) is notparticularly limited, and any condition enabling the formation of thecomplex may be selected. Typical reaction conditions are those used forchemical modification with PEG of the peptide used in the presentinvention.

The reaction conditions are more specifically described in Example 27.

In such production methods, the peptide used in the present invention,the activated PEG, and the peptide-binding substance may be used at anyarbitrary amount and ratio as determined by considering the applicationof the complex, and the properties, characters, pharmacologicalactivity, safety, and the like required for the complex.

Specifically, in step a) of method II), the peptide-binding substance isused such that the ratio of the number of positive charges (+) of thepeptide to the number of negative charges (−) of the peptide-bindingsubstance (±ratio) is 2 or more, preferably 3 or more. The ratio, on theother hand, is preferably up to 100, and more preferably up to 50. Whenthe ratio is within such range, the peptide can form a complex with thepeptide-binding substance.

The activated PEG is used in step b) of method II) at an amount in themolar ratio to the peptide used in the present invention of 0.0001 to10000, preferably 0.01 to 100, and more preferably 0.1 to 10. When theactivated PEG is used at an amount in such a range, the PEG-modifiedpeptide will retain the activity of the original peptide after thereaction between the activated PEG and the peptide, and the PEG-modifiedpeptide will be imparted with favorable properties such as improvedsolubility, reduced antigenicity, and reduced toxicity.

It goes without saying that the complex of the peptide chemicallymodified with PEG of the present invention with the peptide-bindingsubstance, the mixture, composition and aggregates of the threecomponents, and the like are all within the scope of the presentinvention. The results of Example 30 indicate that a complex (aggregate,mixture, composition, or the like) is formed by the peptide used in thepresent invention, the PEG, and the peptide-binding substance arepresent.

EXAMPLES

The present invention is described in further detail by referring to thefollowing Examples which are presented for the purpose of illustrationand which by no means limit the scope of the invention. Theabbreviations used in the following description are those customarilyused in the field of the art.

Example 1 Chemical Synthesis of the Peptide

Peptides having the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO:27 were synthesized by solid phase synthesis by using an automaticpeptide synthesizer (432A, manufactured by Applied Biosystems). It is tobe noted that, in synthesizing a peptide having a length of 30 residuesor longer, synthesis was suspended without deprotecting the 25th aminoacid residue, and the synthesis was accomplished by resuming thesynthesis after providing the synthesizer with the amino acid columns of26th and remaining residues. Unless otherwise noted, the synthesis wasconducted in accordance with the manufacturer's manual. The peptide wascleaved, deprotected, precipitated in ether, stripped of the ether,dissolved in distilled water, and lyophilized. Next, the peptides of SEQID NO: 1 to SEQ ID NO: 26 were dissolved in 20% acetonitrile aqueoussolution containing 10 mM HCl, and the peptide of SEQ ID NO: 27 wasdissolved in 15% acetonitrile/15% isopropanol aqueous solutioncontaining 10 mM HCl. By using C18 column (CAPCELLPAK C18AG120,manufactured by Shiseido) and high performance liquid chromatography(625 LC System, manufactured by Waters), the peptides of SEQ ID NO: 1 toSEQ ID NO: 26 were purified so that single peak is obtained in linearconcentration gradient of 20% to 70% acetonitrile aqueous solutioncontaining 10 mM HCl, and the peptide of SEQ ID NO: 27 was purified sothat single peak is obtained in linear concentration gradient of 15% to50% acetonitrile/15% to 50% isopropanol aqueous solution containing 10mM HCl. The thus purified peptides were lyophilized, dissolved indistilled water, and stored after frozen.

The peptides were produced at a yield of 30 mg to 40 mg, respectively.

Example 2 Assay of Peptide (1)

The resulting synthetic peptides were evaluated for their molecularweight by mass spectroscopy using MALDI-TOFMS mass spectrometer(VoyagerDE-STR, manufactured by PE Biosystems) to thereby confirm thatthe resulting peptides were the desired peptides. Unless otherwisenoted, the spectroscopy was conducted in accordance with themanufacturer's manual. The procedure was as summarized below.

First, α-cyano-4-hydroxycinnamic acid (CHCA) was dissolved in 0.1 vol %TFA/50 vol % acetonitrile/pure water to prepare a 10 mg/mL matrixsolution. Then, 0.5 μL of aqueous solution (10 pmol/μL) of the peptideproduced by the procedure described in Example 1 and 0.5 μL of thematrix solution were mixed on a sample plate, and the mixture was driedfor crystallization of the sample. The analysis was conducted under thefollowing conditions.

Measurement mode: Linear, positive

Calibration: external standard method (Note that the standards were (i)Angiotensin I, (ii) ACTH (1-17clip), (iii) ACTH (18-39clip), (iv) ACTH(7-38clip), and (v) Insulin (bovine)).

It was then confirmed that all of the synthetic and purified peptideswere consistent with the theoretical molecular weight.

Example 3 Assay of Peptide (2)

The solutions of the synthetic peptide produced in Example 1 weredetermined for their concentration by analyzing amino acid compositionby ninhydrine method.

The samples were exsiccated in a glass test tube, and after adding 100μL of 6N HCl and evacuating and sealing the test tube, hydrolysis wasallowed to proceed at 110° C. for 22 hours. The samples were thenexsiccated, dissolved in pure water, and analyzed in an amino acidanalyzer (L-8500, manufactured by HITACHI). The peptide aqueoussolutions had a concentration of 7 to 10 mg/mL.

Example 4 Measurement of Turbidity in BSA

Aqueous solutions were prepared so that the resulting solution had abovine serum albumin (BSA) concentration of 1% and the peptideconcentration of 50 μM. The solutions were evaluated for theirabsorbance at a wavelength of 600 nm by using a spectrophotometer(DU640, manufactured by Beckman). The results are shown in Table 1. Nosample exhibited an absorbance that exceeded 0.1. TABLE 1 SEQ ID NO: OD600 SEQ ID NO: 1 0.03   SEQ ID NO: 2 0.01> SEQ ID NO: 3 0.01> SEQ ID NO:5 0.01> SEQ ID NO: 6 0.01> SEQ ID NO: 7 0.01> SEQ ID NO: 8 0.01> SEQ IDNO: 10 0.01> SEQ ID NO: 11 0.01> SEQ ID NO: 15 0.01> SEQ ID NO: 16 0.01>SEQ ID NO: 17 0.01> SEQ ID NO: 19 0.01> SEQ ID NO: 23 0.01>

Example 5 Measurement of CD Spectrum

The peptide dissolved in 10 mM phosphate buffer (pH=7) containing 50 mMNaCl was evaluated for its CD spectrum in the presence and in theabsence of 5 mM SDS by using a circular dichroism spectrophotometer(J-500A, manufactured by JASCO). The cell length was 1 mm. Themeasurement was conducted at 35° C. for 6 times in total.

FIGS. 7 to 9 are respectively views showing mean residue ellipticity inCD spectroscopy of the peptide of SEQ ID NO: 1, the peptide of SEQ IDNO: 4 and the peptide of SEQ ID NO: 16. As evident in FIGS. 7 to 9, themean residue ellipticity obtained in the presence of SDS was the socalled W curve and local minimums were found in the areas at thewavelength of 205 to 210 nm and 220 to 225 nm, and α-helix structure wasthereby confirmed.

In other words, it was revealed that the peptides of SEQ ID NO: 1, SEQID NO: 4, and SEQ ID NO: 16 showing the gene-introducing ability are inα-helix structure in the presence of SDS while they do not take α-helixstructure in an aqueous solution solely containing an inorganic salt.

Example 6 Synthesis of Oligonucleotide

Unlabeled 21mer oligonucleotide was prepared by purification using OPCcolumn (manufactured by Applied Biosystems). FITC-labeled 21meroligonucleotide was prepared by HPLC using reverse phase chromatography.Synthesis of the oligonucleotide was consigned to Sawady Technology Co.,Ltd.

Example 7 Preparation of Plasmid

For the expression plasmid including firefly luciferase gene as thereporter gene, there were used a commercially available plasmid(pGL3-Control Vector, manufactured by Promega) wherein fireflyluciferase gene had been incorporated under SV40 early promoter, aplasmid (pCMV-Luc(F)) wherein firefly luciferase gene had beenincorporated under CMV early promoter, and a plasmid (pEF-Luc) whereinfirefly luciferase gene had been incorporated under EF-1 α promoter. Forthe expression plasmid including HSV-tk gene as the reporter gene, therewere used a plasmid (pEF-tk) wherein HSV-tk gene had been incorporatedunder EF-1 α promoter, and a plasmid (pCMV-tk) wherein HSV-tk gene hadbeen incorporated under CMV early promoter. These plasmids and pUC119and pBR322 which are respectively a universal plasmid were amplified inE. coli when necessary, and purified by a known method before use.

Example 8 Evaluation of Nucleic Acid-Binding Ability

(1) Evaluation of Binding Ability to Oligonucleotide

The 21mer oligonucleotide prepared by the procedure described in Example6 (final concentration, 6.7 μM) and the peptide prepared by theprocedure described in Examples 1 to 3 were mixed in 20 mM Tris-HClbuffer (pH=7.2) containing 150 mM NaCl at a charge ratio (±ratio) of 0to 10, and the mixture was allowed to stand at 37° C. for 30 minutes.Next, 7.5 μL of this solution was mixed with an equal amount ofTris-borate buffer (pH=8.2) containing 80% formamide, andelectrophoresis was conducted on 25% polyacrylamide gel containing 7Murea. After the electrophoresis, the gel was stained with 5% aqueoussolution of Stains all (manufactured by Funakoshi) containing 50%formamide and washed with water to thereby determine the binding of theoligonucleotide and the peptide.

FIG. 10 shows the electropherogram for the peptide of SEQ ID NO: 1. The±ratio (charge ratio) indicated in FIG. 10 is the ratio of the number(+) of the positively-charged groups in the peptide to the number (−) ofnegatively-charged groups in the nucleic acid. As evident in FIG. 10,the amount of oligonucleotide that became bound to the peptide increasedwith the increase in the charge ratio (±ratio), and the oligonucleotidewas fully bound to the peptide at a charge ratio (±ratio) of 10. Thepeptides of SEQ ID NO: 2 to SEQ ID NO: 24 were also found to bind at acharge ratio (±ratio) of 10.

(2) Evaluation of Binding Ability to Plasmid

The plasmid prepared by the procedure described in Example 7 (pUC119;final concentration, 20 μg/mL) and the peptides prepared by theprocedure described in Examples 1 to 3 were mixed in 20 mM Tris HClbuffer (pH=7.2) containing 150 mM NaCl at a charge ratio (±ratio) of 0to 3, and the mixture was allowed to stand at 37° C. for 30 minutes.Electrophoresis was then conducted on 1% agarose gel to therebydetermine the binding of the plasmid and the peptide.

FIG. 11 shows the electropherogram for the peptide of SEQ ID NO: 1. The±ratio (charge ratio) indicated in FIG. 11 is the ratio of the number(+) of the positively-charged groups in the peptide to the number (−) ofnegatively-charged groups in the plasmid. As evident in FIG. 11, theamount of plasmid that became bound to the peptide increased with theincrease in the charge ratio (±ratio), and the plasmid was fully boundto the peptide at a charge ratio (±ratio) of 3. The peptides of SEQ IDNO: 2 to SEQ ID NO: 24 were also found to bind at a charge ratio(±ratio) of 3.

Example 9 Evaluation of Nucleic Acid-Introducing Ability (1)

A cell line established from monkey kidney (Vero cell) and a humanbladder cancer cell (T24 cell) purchased from American Type CultureCollection (ATCC) and human lung cancer cell (A549 cell) purchased fromDainippon Pharmaceutical were inoculated on a 24 well culture plate(manufactured by NALGEN NUNC) at 1×10⁵ cells/well. Vero cell and A549cell were cultivated in DMEM (manufactured by Life TechnologiesOriental) supplemented with 10% fetal bovine serum (hereinafter referredto as FBS, manufactured by NICHIREI), and T24 cell was cultivated inMcCoy's 5a (manufactured by Life Technologies Oriental) supplementedwith 10% FBS in 5% CO₂ atmosphere at 37° C. for 24 hours. After removingthe medium, opti-MEM (manufactured by Life Technologies Oriental)prepared to have the concentration of the luciferase-expressing plasmidprepared in Example 7 of 1 μg/mL and the concentration of the peptideprepared in Example 1 of 1.25, 2.5, or 5 μM was added, and the cellswere cultivated for 5 hours. Next, in the case of Vero cell and A549cell, the culture medium was replaced with DMEM supplemented with 10%FBS, and in the case of T24 cell, the medium was replaced with McCoy's5a supplemented with 10% FBS, and cultivation was continued in 5% CO₂atmosphere and at 37° C. for another 24 hours. The luciferase activityexpressed in the cell was then measured by the method instructed inluciferase assay system (manufactured by Promega). To be more specific,the cells were washed with phosphate-buffered saline (hereinafterreferred to as PBS, manufactured by SIGMA), and the cells were lyzedwith Passive Lysis Buffer attached to the kit.

20 μL of this cell lysate and 100 μL of the Luciferase Assay Reagent IIattached to the kit was added to a fluorescence-measuring plate(Microlite™ 1 plate, manufactured by Dynatech), and after mixing,fluorescence was measured for 1 second by using a multilabel counter(ARVO™ SY1420 MULTILABEL COUNTER, manufactured by Wallac Beltold.

Concentration of the protein in the cell lysate was measured by mixing 8μL of the cell lysate and 200 μL of the protein assay solution(manufactured by Biorad) with 792 μL of ultrapure water in a disposablecuvette (UV fluorescent cuvette A204X, manufactured by Funakoshi),allowing the mixture to stand for 5 minutes at room temperature, andmeasuring the absorption at 595 nm with a spectrophotometer (DU640,manufactured by Beckman). Bovine serum albumin (BSA) was used for thestandard protein. By using the thus obtained results, count per 1 secondper 1 mg of protein of the cell lysate was calculated to thereby use thecount as the luciferase activity, and the highest count in the threeconditions of different peptide concentration was designated thegene-introducing ability of the peptide.

Example 10 Evaluation of Nucleic Acid-Introducing Ability (2)

Vero cell was inoculated in the well of a chamber slide (Lab-Tek IIchamber slide, size 4 well, manufactured by NALGEN NUNC) at a rate of1×105 cells/well, and the cells were cultivated in DMEM supplementedwith 10% FBS in 5% CO₂ atmosphere at 37° C. for 24 hours. After removingthe medium, opti-MEM containing 300 nM FITC-labeled oligonucleotideprepared in Example 6 and 2.5 μM of the peptide of SEQ ID NO: 1 preparedin Example 1 was added, and the cultivation was continued for 4 hours.The medium was replaced with DMEM supplemented with 10% FBS, and thecultivation was continued in 5% CO₂ atmosphere at 37° C. for another 24hours. After washing the cell with PBS, the cells were fixed by usingPBS containing 4% paraformaldehyde, and accumulation of FITC-labeledoligonucleotide in the cell nucleus was confirmed by using afluorescence microscope (AH-3, manufactured by Olympus). It was thenconfirmed that FITC-labeled oligonucleotide was accumulated in the cellnucleus. On the other hand, accumulation of FITC-labeled oligonucleotidein the cell was not confirmed in the absence of the peptide of SEQ IDNO: 1.

Example 11 Evaluation of Nucleic Acid-Introducing Ability into Cell ofthe Peptide (1)

The peptide of SEQ ID NO: 1 was evaluated for its ability of introducingthe nucleic acid into a cell by the procedure described in Example 9.FIG. 12 is a view wherein difference in the amount of plasmid introducedinto the cell is compared in the presence and absence of the peptide ofSEQ ID NO: 1 by using luciferase activity. As shown in FIG. 12, thepeptide of SEQ ID NO: 1 was found to have the ability of introducing thenucleic acid into a cell, while the nucleic acid was not introduced inthe absence of the peptide of SEQ ID NO: 1.

Example 12 Evaluation of Nucleic Acid-Introducing Ability into Cell ofthe Peptide (2)

The peptides of SEQ ID NO: 2 to SEQ ID NO: 18 having a sequence whereinone or two locations in “the amino acid sequence of 18 amino acidsexhibiting the four sided structure of the present invention” have beensubstituted in the peptide of SEQ ID NO: 1 was evaluated for theirability of introducing the nucleic acid into a cell by the proceduredescribed in Example 9. It was then found that all peptides had theability of introducing the nucleic acid into a cell.

It is to be noted that the nucleic acid-introducing ability wasclassified as described below on the bases of the fluorescent count per1 mg of protein per 1 second (cps/mg protein). The results are listed inTable 2. 10,000 to less than 100,000 1+ 100,000 to less than 1,000,0002+ 1,000,000 to less than 10,000,000 3+ 10,000,000 to less than100,000,000 4+ 100,000,000 to less than 1,000,000,000 5+ 1,000,000,000or more 6+

It is to be noted that the nucleic acid-introducing ability of SEQ IDNO:1 was 3+ in the above classification. TABLE 2 Nucleic acid-introducing SEQ ID NO: ability SEQ ID NO: 2 3+ SEQ ID NO: 3 2+ SEQ IDNO: 4 4+ SEQ ID NO: 5 2+ SEQ ID NO: 6 2+ SEQ ID NO: 7 2+ SEQ ID NO: 8 3+SEQ ID NO: 9 3+ SEQ ID NO: 10 3+ SEQ ID NO: 11 3+ SEQ ID NO: 12 2+ SEQID NO: 13 3+ SEQ ID NO: 14 3+ SEQ ID NO: 15 2+ SEQ ID NO: 16 4+ SEQ IDNO: 17 2+ SEQ ID NO: 18 4+ SEQ ID NO: 19 4+ SEQ ID NO: 20 4+ SEQ ID NO:21 2+ SEQ ID NO: 22 3+ SEQ ID NO: 23 2+ SEQ ID NO: 24 1+

Example 13 Evaluation of Nucleic Acid-Introducing Ability into Cell ofthe Peptide (3)

The peptides of SEQ ID NO: 19 to SEQ ID NO: 22 having a sequence whereinthree locations in “the amino acid sequence of 18 amino acids exhibitingthe four sided structure of the present invention” had been substitutedin the peptide of SEQ ID NO: 1 was evaluated for their ability ofintroducing the nucleic acid into a cell by the procedure described inExample 9. It was then found that these peptides had the ability ofintroducing the nucleic acid into a cell.

It is to be noted that the nucleic acid-introducing ability wasclassified as described in Example 12 on the bases of the fluorescentcount per 1 mg of protein per 1 second. The results are listed in Table2.

Example 14 Evaluation of Nucleic Acid-Introducing Ability into Cell ofthe Peptide (4)

The peptides of SEQ ID NO: 23 and SEQ ID NO: 24 which are respectively adeletion mutant of the peptides of SEQ ID NO: 1 and SEQ ID NO: 4 wereprepared, and these peptides were evaluated for their ability ofintroducing the nucleic acid into a cell by the procedure described inExample 9. It was then found that all of the peptides had the ability ofintroducing the nucleic acid into a cell.

It is to be noted that the nucleic acid-introducing ability wasclassified as described in Example 12 on the bases of the fluorescentcount per 1 mg of protein per 1 second. The results are listed in Table2.

Example 15 Evaluation of Nucleic Acid-Introducing Ability into Cell ofthe Peptide (5)

The peptide of SEQ ID NO: 16 was evaluated for its ability ofintroducing the nucleic acid into various cancer cell lines by theprocedure described in Example 9.

The cancer cell used were human uterine cancer cell, MES-SA/Dx5(purchased from ATCC), a transformant cell from human kidney 293(purchased from ATCC), human hepatoma cell, SK-HEP-1 (purchased fromATCC), human ovarian cancer cell, SK-OV-3 (purchased from ATCC), ratbrain tumor cell, F98 (purchased from ATCC), mouse melanoma cell,B16/BL6 (provided by Chemistry Division, Institute of ImmunologicalScience, Hokkaido University), human uterine cancer cell, MES-SA(purchased from ATCC), mouse lung cancer cell, Lewis Lung Carcinoma(provided by Japanese Foundation for Cancer Research), mouse hapatomacell, MH134 (provided by Central Laboratories for Experimental Animals),human uterine cancer cell, MES-SA/Mx2 (purchased from ATCC), humanmedulloblastoma cell, Daoy (purchased from ATCC), human uterine cervixcancer cell, HeLa (purchased from ATCC), human breast cancer cell, MCF7(purchased from ATCC), human glioblastoma cell, U-87 MG (purchased fromATCC), human breast cancer cell, MDA-MB-468 (purchased from ATCC), humanrenal cancer cell, A-498 (purchased from ATCC), mouse melanoma cell,B16/F10 (purchased from ATCC), human prostatic cancer cell, DU 145(purchased from ATCC), human brain tumor cell, U-138.MG (purchased fromATCC) and mouse sarcoma cell, Meth-A (provided by National CancerCenter). The culture media used for the propagation of the cells areindicated in Table 5. It is to be noted that, of the additives, NEAA(manufactured by ICN) is a nonessential amino acid, Na-Pyr (manufacturedby ICN) is sodium pyruvate. The FBS used were the one manufactured byNICHIREI in all cases, and the culture media were those manufactured byLife Technologies Oriental. All propagation media were supplemented withpenicillin/streptomycin (manufactured by ICN).

Subcultured cells were inoculated on 24 well culture plate at a rate of1×10⁵ cells/well, and the cells were cultivated in the respectiveculture medium in 5% CO₂ atmosphere at 37° C. for 24 hours. Afterremoving the medium, opti-MEM prepared such that theluciferase-expressing plasmid concentration prepared in Example 7 was 1μg/mL and the peptide concentration prepared in Example 1 was 2.5 μM wasadded, and the cultivation was continued for 5 hours. The medium wasthen replaced with the respective propagation medium, and thecultivation was continued in 5% CO₂ atmosphere at 37° C. for another 24hours. Luciferase activity expressed in each cell was then measured bythe procedure described in Example 9, and the gene introduction abilityof the peptides is indicated in Table 3 in accordance with the criteriadescribed in Example 12. TABLE 3 Nucleic acid- introducing Cell lineTissue Medium used for propagation ability MES-SA/Dx5 human uterinecancer McCoy's 5A supplemented with 10% FBS 6+ 293 human kidney DMEMsupplemented with 10% FBS 6+ SK-HEP-1 human hepatoma MEM supplementedwith 10% inactivated FBS 5+ (NEAA, Na, Pyr added) SK-OV-3 human ovariancancer RPMI1640 supplemented with 15% FBS 5+ F98 rat brain tumor DMEMsupplemented with 10% FBS 5+ B16/BL6 mouse melanoma MEM supplementedwith 10% inactivated FBS 5+ (NEAA, Na, Pyr added) MES-SA human uterinecancer McCoy's 5A supplemented with 10% FBS 5+ Lewis Lung mouse lungcancer DMEM supplemented with 10% FBS 4+ Carcinoma MH-134 mouse hepatomaRPMI1640 supplemented with 10% inactivated FBS 4+ MES-SA/Mx2 humanuterine cancer 1:1 mixture of Waymouth's MB752 and McCoy's 5A 4+supplemented with 10% FBS Daoy human medulloblastoma MEM supplementedwith 10% FBS 4+ HeLa human uterine cervix DMEM supplemented with 10% FBS4+ cancer MCF7 human breast cancer RPMI1640 supplemented with 10%inactivated FBS 4+ U-87 MG human glioblastoma MEM supplemented with 10%inactivated FBS 4+ (NEAA, Na, Pyr added) MDA-MB-468 human breast cancerRPMI1640 supplemented with 10% inactivated FBS 4+ A-498 human renalcancer DMEM supplemented with 10% FBS 4+ B16/F10 mouse melanoma DMEMsupplemented with 10% FBS 4+ DU 145 human prostatic cancer MEMsupplemented with 10% inactivated FBS 4+ (NEAA, Na, Pyr added) U-138 MGhuman brain tumor DMEM supplemented with 10% FBS 4+ Meth-A mouse sarcomaDMEM supplemented with 10% FBS 4+

Example 16 Evaluation of Nucleic Acid-Introducing Ability into Cell ofthe Peptide (6)

The HSV-tk-expressing plasmid prepared in Example 7 was introduced inLewis lung carcinoma cell (mouse lung cancer cell) by using the peptideof SEQ ID NO: 16 prepared in Example 1 to thereby evaluate the effect ofincreasing the sensitivity for ganciclovir (hereinafter referred to asGCV). To be more specific, cells were inoculated in 96 well cultureplate (manufactured by NALGEN NUNC) at a rate of 5×10³ cells/well, andcultivated in DMEM supplemented with 10% FBS in 5% CO₂ atmosphere at 37°C. for 24 hours. After removing the culture medium, 100 μL of opti-MEMprepared to have a concentration of the HSV-tk-expressing plasmid of 1μg/mL and a concentration of the peptide of SEQ ID NO: 16 of 2.5 μM wasadded to each well, and the cultivation was continued for 5 hours. Next,100 μL of the DMEM supplemented with 20% FBS respectively containing 0,2, 20, and 200 μM of GCV (Denosine, manufactured by Tanabe Seiyaku) wasadded to each well, and cultivation was continued in 5% CO₂ atmosphereat 37° C. for 3 days. The effect of suppressing the cell propagation wasmeasured by WST-1 assay by adding 10 μL of WST-1 solution (manufacturedby Takara Shuzo) in each well, allowing the reaction to proceed for 1hour, and measuring the absorbance at a wavelength of 620 nm with amulti label counter by using a reference wavelength of 450 nm. As shownin FIG. 13, the cell propagation was found to be suppressed in the caseof the Lewis lung carcinoma cell having the HSV-tk-expressing plasmidintroduced therein in a manner dependent on the dose of the GCV whereasthe effect of increasing the GCV sensitivity was not found in thecontrol cell having the luciferase-expressing plasmid introducedtherein.

Example 17 Evaluation of Storage Stability of the Complex with NucleicAcid

Storage stability of the complex of the plasmid and the peptide wasevaluated.

Luciferase-expressing plasmid was introduced in Vero cell in accordancewith the procedure described in Example 9 by using the opti-MEM preparedto have a concentration of the luciferase-expressing plasmid prepared inExample 7 of 2 μg/mL and a concentration of the peptide of SEQ ID NO: 4prepared in Example 1 of 5 μM to thereby measure the luciferaseactivity. The opti-MEM containing plasmid and peptide used were the onewhich had been prepared immediately before the gene introduction, andthe one which had been prepared 1 week (7 days) in advance and stored at4° C.

FIG. 14 shows gene-introducing activity of the peptide/plasmid complexstored at 4° C. for 1 week as a value in relation to thegene-introducing activity of the peptide/plasmid complex preparedimmediately before the gene introduction which is assumed to be 100%. Asshown in FIG. 14, the gene-introducing activity of the peptide/plasmidcomplex stored at 4° C. for 1 week was substantially equivalent to thatof the gene-introducing activity of the peptide/plasmid complex preparedimmediately before the gene introduction.

For the purpose of reference, gene-introducing activity of Lipofectinand LipofectAMINE 2000 (manufactured by Life Technologies Oriental),which are commercially available plasmid-introducing agents, were alsoevaluated by mixing these reagent with luciferase-expressing plasmid inaccordance with the attached protocol, and evaluating theirgene-introducing activity after storing at 4° C. for 1 week. BothLipofectin and LipofectAMINE 2000 exhibited a marked decrease in thegene-introducing activity. The results are also shown in FIG. 14.

Example 18 Evaluation of Nuclease Resistance-Imparting Ability (1)

Nuclease resistance-imparting ability for natural (P═O form)oligonucleotide was evaluated. To be more specific, 2 U of nuclease Bal31 (manufactured by Takara Shuzo) was added to 20 mM Tris-HCl buffer(pH=8) containing 50 μM of the peptide of SEQ ID NO: 1 prepared inExample 1, 3 μM of P═O form oligonucleotide prepared in Example 6, 150mM NaCl, 12 mM MgCl₂, and 12 mM CaC₁₂, and the reaction was allowed totake place at 30° C. for 60 minutes. Next, the oligonucleotide whichescaped from the decomposition was extracted with phenol/chloroform, andsubjected to electrophoresis by the procedure described in Example 8 tostain the oligonucleotide.

FIG. 15 shows the electropherogram. As shown in FIG. 15, the peptide ofSEQ ID NO: 1 was found to exhibit nuclease resistance-imparting abilityfor the natural (P═O form) oligonucleotide.

For the purpose of reference, nuclease resistance-imparting ability wasalso evaluated for lipofectin and LipofectAMINE 2000, which arecommercially available plasmid-introducing agents. Both lipofectin andLipofectAMINE 2000 exhibited no stained oligonucleotide in theelectropherogram, indicating the decomposition of the oligonucleotide.The results are shown in FIG. 15.

Example 19 Evaluation of Nuclease Resistance-Imparting Ability (2)

pBR322 prepared by the procedure described in Example 7 (finalconcentration, 20 μg/mL) and the peptide of SEQ ID NO: 16 prepared inExample 1 were mixed in 20 mM Tris-HCl buffer (pH=8) containing 150 mMNaCl and 1.7 mM MgCl₂ at a charge ratio (±ratio) of 0 to 10, and 5 U ofDNase I (manufactured by Takara Shuzo) was then added. After allowingthe reaction to proceed at 30° C. for 60 minutes, the plasmid whichescaped the decomposition was extracted with phenol/chloroform, andsubjected to electrophoresis by the procedure described in Example 8 tostain the plasmid.

FIG. 16 shows the electropherogram. As shown in FIG. 16, the peptide ofSEQ ID NO: 16 was found to exhibit nuclease resistance-imparting abilityfor the plasmid.

Example 20 Evaluation of the Affinity of the Peptide for PhosphatidylSerine (EIA)

The specific affinity of the peptide for phosphatidyl serine wasevaluated by measuring activity of the peptide for inhibiting thebinding of the human Factor VIII to phosphatidyl serine by using humanFactor VIII which is known to bind to phosphatidyl serine but not tophosphatidyl choline in the presence of serum albumin. To be morespecific, 100 μL of ethanol solution containing 10 μg/mL ofphospholipids at a phosphatidyl serine (manufactured by SIGMA):phosphatidyl choline (manufactured by SIGMA) ratio of 3:7 was added tothe wells of a 96 well plate (Immulon I, manufactured by Dynatech), andexsiccated by using a centrifugal evaporator (EC-95C, manufactured bySakuma Seisakusho) at 40° C. for 40 minutes. 200 μL each of TBS (10 mMTris-HCl (pH 7.4) containing 1% bovine serum albumin (BSA Fraction V,hereinafter referred to as BSA, manufactured by Seikagaku Corporation)and 0.9% (W/V) NaCl solution were added to each well, and the wells wereblocked by allowing the plate to stand at 37° C. for 2.5 hours. Afterwashing the plate with water, 100 μL of TBS solution supplemented with5% BSA mixed with human Factor VIII (manufactured by AmericanDiagnostica) at a final concentration 1 μg/mL and the peptide ofpredetermined dose prepared in Example 1 was added to each well, and thereaction was allowed to take place at 4° C. for 24 hours.

After the completion of the reaction, the human Factor VIII that becamebound to the phosphatidyl serine was measured by enzyme immunoassay(EIA) in accordance with the general procedure described in a book(“Enzyme Immunoassay (3rd ed.)”, Eiji Ishikawa et al., Igaku-Shoin,1987). To be more specific, the measurement was conducted by usinganti-human Factor VIII mouse monoclonal antibody (ESH8, manufactured byAmerican Diagnostica) for the primary antibody, horseradishperoxidase-labeled anti-mouse IgG antibody (P0260, manufactured by Daco)for the secondary antibody, and tetramethylbenzidine for the chromogenicsubstrate, and measuring the absorption at a wavelength of 450 nm byusing a reference wavelength of 630 nm on a spectrophotometer (NJ-2100,manufactured by Intermed).

Intensity of the affinity of the peptide for the phosphatidyl serine wasevaluated by using the value obtained by subtracting the absorption ofthe well having no human Factor VIII added thereto from the absorptionof the well having only the human Factor VIII added thereto as thecontrol value (100%), and designating the peptide concentration at whichthe value 0.5 was obtained when the value obtained by subtracting theabsorption of the well having only the peptide added thereto from theabsorption of the well having a mixture of the human Factor VIII and thepeptide added thereto were divided by the control value as the IC₅₀value, and evaluating the intensity to be ++ when the IC₅₀ value was upto 1 μM, and + when the intensity was more than 1 μM but not more than10 μM. It is to be noted that the specific affinity was evaluated to benone when the intensity was 10 μM or more. The results are shown inTable 4. TABLE 4 Affinity for SEQ ID NO: phosphatidyl serine SEQ ID NO:1 ++ SEQ ID NO: 2 ++ SEQ ID NO: 3 ++ SEQ ID NO: 5 ++ SEQ ID NO: 6 ++ SEQID NO: 7 + SEQ ID NO: 8 + SEQ ID NO: 10 + SEQ ID NO: 11 + SEQ ID NO: 15++ SEQ ID NO: 16 ++ SEQ ID NO: 17 ++ SEQ ID NO: 19 ++ SEQ ID NO: 23 ++

Example 21 Evaluation of the Affinity of the Peptide for PhosphatidylSerine (2)

The specific affinity of the peptide for phosphatidyl serine wasevaluated by means of surface plasmon resonance (SPR) using Biacore 2000(manufactured by Biacore). To be more specific, phosphatidyl choline wasimmobilized on flow cell 1, and 50% of phosphatidyl serine and 50% ofphosphatidyl choline were immobilized on flow cell 2 of HPA chip(manufactured by Biacore), and ability of the peptide to bind to thephospholipid was evaluated in the presence of 0.1 mg/mL BSA to therebyevaluate the affinity. It was then found that the binding of the peptideSEQ ID NO: 1 to the flow cell 2 having 50% of phosphatidyl serine and50% of phosphatidyl choline immobilized thereto was stronger than thatof the flow cell 1 having phosphatidyl choline immobilized thereto, andthe affinity of the peptide specific for the phosphatidyl serine wasthereby indicated (FIG. 17). In contrast, the peptide of SEQ ID NO: 25wherein the sequence of SEQ ID NO: 1 had been randomized showed nobinding to neither the flow cell 1 having phosphatidyl cholineimmobilized thereto nor the flow cell 2 having 50% of phosphatidylserine and 50% of phosphatidyl choline immobilized thereto, indicatingthe absence of the affinity (FIG. 18).

Example 22 Correlation Between the Amount of Phosphatidyl SerineTranslocation and the Introduction Efficiency (Selectivity)

In order to elucidate the correlation between the level of thegene-introducing ability of the peptide and the affinity forphosphatidyl serine, an investigation was conducted by using cellsexhibiting different amount of phosphatidyl serine translocation to thecell surface. To be more specific, the cells used were Vero, A549, andT24 cells, and the luciferase-expressing plasmid prepared in Example 7was introduced in the cells by using the peptide of SEQ ID NO: 1prepared in Example 1 in accordance with the description of Example 9 tothereby measure luciferase activity. In the meanwhile, the amount ofphosphatidyl serine translocated to the outer surface of the cellmembrane of the cells was measured by labeling the cells withFITC-labeled ANNEXIN V (ANNEXIN V-FITC, manufactured by Pharmingen) inaccordance with the manual attached thereto, and conducting flowcytometry (FACS Calibur, manufactured by Becton Dickinson). To be morespecific, the cells that had been scraped from the culture flask weremixed with the solution of the FITC-labeled ANNEXIN V, and the cellswere measured for their FITC fluorescent intensity by flow cytometry.Average fluorescent intensity of each cell was then designated theamount of phosphatidyl serine translocation for each cell line.

Correlation was then found between the amount of phosphatidyl serinetranslocation (amount of ANNEXIN V binding) and the gene introductionactivity of the peptide vector as shown in Table 5.

For the purpose of reference, the procedure as described above wasrepeated by using lipofectin (manufactured by Life TechnologiesOriental) which is a commercially available plasmid-introducing agent inaccordance with the attached protocol. It was then found that theplasmid was equally introduced in every type of cells, indicating theabsence of the specific recognition of the phosphatidyl serine. TABLE 5Luciferase activity (cps/mg protein) Amount of Peptide Cell Annexin-Vbound (SEQ ID NO: 1) Lipofectin Vero 165 2.5 × 10⁶ 1.1 × 10⁶ A549 82 8.7× 10⁴ 8.4 × 10⁵ T24 32 7.8 × 10³ 1.1 × 10⁶

Example 23 Correlation Between the Amount of Phosphatidyl SerineTranslocation and the Introduction Efficiency (2)

In order to elucidate the correlation between the gene-introducingability of the peptide and the affinity for the phosphatidyl serine,nucleic acid-introducing ability of the peptide was investigated byusing the cells wherein the phosphatidyl serine had not beentranslocated, and the cells wherein the phosphatidyl serine has beentranslocated by stimulating the cell. The cell used was RBL-2H3 cellfrom rat basophil (purchased from ATCC), and this cell was sensitizedwith anti-DNP mouse monoclonal IgE antibody (manufactured by SIGMA) anddegranulated with DNP-BSA (manufactured by Calbiochem) Luciferase genewas introduced to such cell by using the peptide of SEQ ID NO: 16 inaccordance with the procedure described in Example 9 to thereby measurethe luciferase activity. To be more specific, RBL-2H3 cells wereinoculated in a 24 well plate at a rate of 3×10⁵ cells/well, and afteradding anti-DNP mouse monoclonal IgE antibody to a concentration of 100ng/mL, the cells were cultivated for 24 hours. After washing the cellstwice with PBS, DNP-BSA was added to 10 ng/mL to cause degranulation for45 minutes. After removing the culture medium and washing the well withphysiological saline, opti-MEM having a concentration of theluciferase-expressing plasmid prepared in Example 7 of 1 μg/mL and aconcentration of the peptide of SEQ ID NO: 16 prepared in Example 1 of2.5 μM was added, and cultivation was continued for 5 hours.

The medium was then replaced with MEM supplemented with 15% inactivatedFBS (having NEAA and Na.Pyr added thereto), and incubation was continuedin 5% CO₂ atmosphere at 37° C. for 1 day. The cells were then scrapedoff, and evaluated for their luciferase activity by the proceduredescribed in Example 9. In the meanwhile, amount of phosphatidyl serinetranslocated in the RBL-2H3 cell by degranulation was measured by theprocedure as described below, namely, by inoculating the RBL-2H3 cell ina 6 well culture plate (manufactured by NALGEN NUNC) at a rate of1.5×10⁶ cells per well, adding anti-DNP mouse monoclonal IgE antibody toa concentration of 100 ng/mL, and cultivating in MEM supplemented with15% inactivated FBS (having NEAA and Na-Pyr added thereto) in 5% CO₂atmosphere at 37° C. for 24 hours. After washing the wells twice withPBS, DNP-BSA was added to 10 ng/mL to thereby cause degranulation for 45minutes. After scraping off the cells, the cells were labeled withFITC-labeled ANNEXIN V (manufactured by MBL) in accordance with theprocedure described in the attached manual, and the activity wasmeasured by flow cytometry.

It was then found that the cells stimulated for degranulation had thephosphatidyl serine translocated to its surface (FIG. 19), and thegene-introducing ability of the peptide into the RBL-2H3 cell that hadbeen stimulated for degranulation was significantly high compared to thecase of the undegranulated cell (FIG. 20).

It is to be noted that the gene-introducing ability of LipofectAMINE2000 which is a commercially available gene-introducing agent wasequivalent or slightly lower in the case of the degranulated cellcompared to the case of the cell before the degranulation.

Example 24 Evaluation of Nucleic Acid-Introducing Ability into Cell ofthe Peptide (7)

In order to demonstrate the in vivo gene-introducing ability of thepeptide, nucleic acid-introducing ability of the peptide was examined byusing an ascites cancer model animal having Meth-A mouse sarcoma cellstransplanted thereto. To be more specific, 4×10⁶ Meth-A cells weretransplanted in the abdominal cavity of BALB/c mouse (purchased fromCharles River JAPAN), and after 4 days, a mixture of 30 μg of theluciferase-expressing plasmid prepared in Example 7 and 113 nmol of thepeptide of SEQ ID NO: 16 prepared in Example 1 was administered to theabdominal cavity of the animal. The mouse was killed after another 1day, and Meth-A cell in the abdominal cavity was recovered to therebymeasure the luciferase activity.

It was then found that, as shown in FIG. 21, while no expression ofluciferase was found in the contrast mouse which had been administeredsolely with physiological saline or in the contrast mouse which had beenadministered only with 30 [g luciferase-expressing plasmid, luciferasewas found to be expressed in the mouse which had been administered witha mixture of 30 μg of the luciferase-expressing plasmid and 113 nmol ofthe peptide of SEQ ID NO: 16 to its abdominal cavity.

Example 25 Evaluation of Nucleic Acid-Introducing Ability into Cell ofthe Peptide (8)

In order to demonstrate the pharmacological effects of the in vivo geneintroduction by using the peptide, anti tumor action realized by theincrease of GCV sensitivity by the introduction of HSV-tk gene wasexamined by using the model animal having Lewis lung carcinoma cell(mouse lung cancer cell) inoculated in its abdominal cavity. To be morespecific, 1×10⁵ Lewis lung carcinoma cells were transplanted in theabdominal cavity of a C57BL/6 mouse (purchased from Charles RiverJAPAN), and a mixture of 10 μg of the HSV-tk-expressing plasmid preparedin Example 7 and 40 nmol of the peptide of SEQ ID NO: 16 prepared inExample 1 was administered in the abdominal cavity after the celltransplantation. The mouse was also administered with GCV at a dose of30 mg/kg/day from 1st to 8th day after the transplantation. As shown inFIG. 22, it was then found that while the average survival period of themice was 14 days both in the case of the group administered with thephysiological saline and in the case of the group administered only withGCV at a dose of 30 mg/kg/day, all mice were alive 20 days after thecell transplantation in the case of the group administered with themixture of 10 μg of the HSV-tk-expressing plasmid and 40 nmol of thepeptide of SEQ ID NO: 16 followed by the administration of the GCV at adose of 30 mg/kg/day.

Example 26 Evaluation of Toxicity of the Peptide

The peptide used in the present invention was administered to a mousefrom its tail vein to thereby evaluate its toxicity. The mouse used wasa female BALB/c mouse of 5 week old, and the peptide was used at a doseof 5 mg/kg. The peptide (SEQ ID NO: 16) used in the present inventionwas found to induce no significant effects in the mouse, demonstratingthat no aggregates were formed in the blood by the peptide of thepresent invention, and that the peptide of the present invention ishighly safe.

Example 27 Modification with PEG of the Plasmid/Peptide Complex

A complex of a plasmid and the peptide was modified with activatedpolyethylene glycol (PEG).

First, the PEG-modified plasmid/peptide complex for use in the in vitrogene introduction was prepared by the procedure as described below.

100 μL of physiological saline containing a plasmid including luciferasegene (pCMV-Luc) at a concentration of 16 μg/mL and 100 μL ofphysiological saline containing the peptide of SEQ ID NO: 16 at aconcentration of 48 μM were preliminarily mixed at room temperature.

To this mixture was added 10 μL of physiological saline containingmPEG-SPA5000 (succinimidyl ester of methoxy poly(ethyleneglycol)propionic acid, Average M. W. 5000, manufactured by Shearwater)at a concentration of 23 mg/mL, and the reaction was allowed to proceedat room temperature for 1 hour to produce the PEG-modifiedpCMV-Luc/peptide complex (PEG1).

Next, PEG-modified plasmid/peptide complex for use in the in vivo geneintroduction was prepared by the procedure as described below.

1050 μL of 5% glucose solution containing the plasmid (pCMV-Luc) asdescribed above at a concentration of 268 pg/mL and 1050 μL of 5%glucose solution containing the peptide of SEQ ID NO: 16 at aconcentration of 804 μM was preliminarily mixed at room temperature toform a complex.

To this mixture was added 150 μL of 5% glucose solution containing themPEG-SPA5000 as described above at a concentration of 263.8 mg/mL, andreaction was allowed to proceed at room temperature for 1 hour to obtainPEG-modified pCMV-Luc/peptide complex (PEG2).

Example 28 Evaluation of Gene Introduction Ability of the PEG-modifiedPlasmid/Peptide Complex (in vitro)

The pCMV-Luc/peptide complex (PEG1) produced by the procedure describedin Example 27 or the complex of pCMV-Luc and the peptide of SEQ ID NO:16 (PEG-undmodified complex) was introduced in Vero cell at a plasmidconcentration of 1 μg/mL by the procedure described in Example 9 tomeasure the luciferase activity expressed.

As demonstrated by the results shown in FIG. 23, the luciferase activityof the Vero cell having the PEG-modified complex introduced wasequivalent to the luciferase activity of the Vero cell having thePEG-unmodified complex introduced, confirming that the gene introductionability of the plasmid/peptide complex is not reduced by the PEGmodification.

Example 29 Evaluation of Gene Introduction Ability of the PEG-modifiedPlasmid/Peptide Complex (in vivo)

Gene introduction ability of the PEG-modified plasmid/peptide complexwas evaluated in vivo by using anaphylaxis shock mouse.

First, mice were sensitized with ovalbumin (hereinafter referred to asOVA) by the procedure as described below to obtain anaphylaxis shockmice. OVA (Egg Alubumin, 5× Cryst, manufactured by SeikagakuCorporation) was first adjusted to 32 μg/mL by using 1.8% solution ofsodium chloride.

Next, aluminum hydroxide gel (Alu-Gel-S, manufactured by SERVA) wasadjusted to 8 mg/mL with ultrapure water, and this solution was cooledon ice. To this solution was mixed an equal volume of the OVA solutionas described above with stirring to prepare OVA/aluminum hydroxide gelsolution.

500 μL of this OVA/aluminum hydroxide gel solution was then administeredto abdominal cavity of BALB/c mouse (male, 4 week old, Japan CharlesRiver) for sensitization with the OVA. The sensitization was conductedtwice at an interval of 5 days.

The gene introduction was conducted on the 13th day after the start ofthe sensitization.

To be more specific, the PEG-modified pCMV-Luc/peptide complex (PEG2)produced by the procedure described in Example 27 or the(PEG-undmodified) complex of pCMV-Luc and the peptide of SEQ ID NO: 16was systemically administered to the mice that had been sensitized withOVA as described above from their tail vein at a dose of 50 μgcalculated in terms of pCMV-Luc simultaneously with 50 μg of OVA.

Right lung was extirpated the next day, and the extirpated lung washomogenized in 500 μL of Lysis Buffer (manufactured by Promega) usingHandy Pestle (manufactured by Toyobo). The lysate was centrifuged by asmall-size centrifuge (manufactured by Tomy) at 12000 rpm for 15minutes, and luciferase activity was measured for 20 μL of thesupernatant by the procedure of Example 9.

As demonstrated in the results shown in FIG. 24, the luciferase activitywas clearly higher in the lung of the mouse administered with thePEG-modified complex compared to that in the lung of the mouseadministered with the corresponding PEG-unmodified complex.

Example 30 Identification of PEG-modified Peptide in the PEG-modifiedPlasmid/Peptide Complex

550 μL of the reaction solution of the pCMV-Luc/peptide complex (PEG1)prepared in Example 27 was charged in a centrifugal ultrafiltrationdevice (CentriconYM-100, manufactured by Amicon), and centrifuged at 4°C. and 1,000×g for 10 minutes to separate and remove the free peptideand the free PEG which had not reacted, and the step of adding 500 μL ofphysiological saline and centrifuging at 4° C. and 1,000×g for 10minutes was repeated for another 3 times. After washing, 50 μL ofnon-reduced SDS sample buffer (manufactured by Daiichi Pure Chemicals)was added, and the reaction system was allowed to stand at roomtemperature for 5 minutes to collect the sample solution (M) containingpCMV-Luc/peptide complex (PEG1) remaining on the ultrafiltrationmembrane.

Next, 25 μL of this sample solution (M) was subjected to SDSpolyacrylamide gel electrophoresis (constant current of 20 mA, 90minutes) by using 5 to 20% gradient gel (manufactured by Atto). The gelafter the electrophoresis was stained by using Silver Staining Kit II(manufactured by WakoPure Chemicals).

As a consequence, a broad stained image was found in the vicinity of themolecular weight of 10,000 Da to 20,000 Da as shown in FIG. 25, andsince the molecular weight of the peptide and the molecular weight ofthe PEG per 1 molecule are about 5,000 Da, respectively, the number ofthe PEG molecules bonded to 1 peptide molecule was deduced to be 1 to 3.

Example 31 Chemical Synthesis and Confirmation of the Peptide used forModification

Peptides having cysteine attached to the C terminal or N terminal of thepeptide of SEQ ID NO: 16 (hereinafter referred to as peptide 16CC andpeptide 16NC respectively) were synthesized by solid phase synthesis byusing an automatic peptide synthesizer (model 433 manufactured byApplied Biosystems) in accordance with the manufacturer's manual. Thepeptide was cleaved, deprotected, precipitated in ether, stripped of theether, dissolved in distilled water, and lyophilized. Next, the peptidewas dissolved in 20% acetonitrile aqueous solution containing lOmM HCl.By using C18 column (CAPCELLPAK C18AG120, manufactured by Shiseido) andhigh performance liquid chromatography (625 LC System, manufactured byWaters), the peptide was obtained in linear concentration gradient of20% to 70% acetonitrile aqueous solution containing 10 mM HCl. The thuspurified peptides were lyophilized, dissolved in distilled water, andstored after lyophilization. The yield was 40 mg to 50 mg, respectively.

Next, the thus obtained peptide was confirmed that it was the peptidedesired by the procedure described in Example 2, and the peptideconcentration was determined by the procedure described in Example 3.

Example 32 Preparation of the Peptide Chemically Modified with PEGhaving a Phospholipid Bonded Thereto

“Peptides chemically modified with PEG having a phospholipid bondedthereto” were prepared by bonding peptide 16CC or peptide 16NC on theterminal of the PEG moiety having maleimide group on its end andsynthetic phospholipid added thereto (DSPE-20MA, manufactured by NOFcorporation) (hereinafter referred to as DSPE-16CC and DSPE-16NC,respectively). More specifically, an aqueous solution of DSPE-20MA at 6μmol/mL was prepared, and aqueous solutions of peptide 16CC and aqueoussolutions of peptide 16NC of 150 nmol/mL, 300 nmol/mL, and 600 nmol/mLwere prepared. Next, 30 μL of DSPE-20MA solution and 30 μL of peptidesolutions of different concentration were mixed in equal amount, and themixtures were allowed to react at room temperature for 2 hours topromote conversion of 2.5%, 5%, and 10% of the DSPE-20MA to DSPE-16CC orDSPE-16NC. The reaction was then terminated by adding 30 μL of 18μmol/mL aqueous solution of cysteine. A control was prepared by adding30 μL of water instead of the peptide solution.

Example 33 Preparation of Doxorubicin-Containing Liposome Modified withthe Peptide Chemically Modified with PEG

A liposome preparation modified with the peptide chemically modifiedwith PEG was prepared. First, a doxorubicin-containing liposome wasproduced using negatively charged liposome (EL-A-01, manufactured by NOFcorporation) and doxorubicin hydrochloride (manufactured by Wako PureChemical) by suspending EL-A-01 in 5% glucose solution at 50 μmol/mL andsuspending doxorubicin hydrochloride in 5% glucose solution at 5μmol/mL, mixing 30 μL of the EL-A-01 suspension and 30 μL of thedoxorubicin hydrochloride solution at equal amount, and heating themixture to 60° C. for 5 minutes. The DSPE-16CC or the DSPE-16NC preparedin Example 32 was fused to the surface of the thus produceddoxorubicin-containing liposome by mixing the solution of thedoxorubicin-containing liposome (60 μL) with 75 μL of the DSPE-16CCsolution or the DSPE-16NC solution prepared in Example 32, and heatingthe mixture to 60° C. for 5 minutes.

The reaction mixture was centrifuged (10,000×g, 20 minutes, 4° C.), andthe supernatant was removed to remove the doxorubicin that had not beenincorporated in the liposome. After repeating this procedure once, thecentrifugate was suspended in 120 μL of 5% glucose solution to obtaindoxorubicin-containing liposome modified with the peptide chemicallymodified with PEG.

Example 34 Quantitation of Doxorubicin in the Doxorubicin-ContainingLiposome Modified with the Peptide Chemically Modified with PEG

Concentration of the doxorubicin in the liposome prepared in Example 33was quantitatively measured by mixing 20 μL of the solution of theliposome prepared in Example 33 and 180 μL of 5% glucose solution,diluting 10 times, further adding 400 μL of isopropyl alcohol to themixture to lyse the liposome, and measuring absorption of the solutionat 470 nm to determine the amount of doxorubicin released. In themeanwhile, calibration curve was depicted by preparing 5% glucosesolutions containing 0, 12.5, 25, 50, and 100 μg/mL of doxorubicin foruse as standard samples, and mixing 200 ml of this solution with 400 μLof the isopropyl alcohol.

Example 35 Evaluation of the Doxorubicin-Containing Liposome Modifiedwith the Peptide Chemically Modified with PEG for its Effect onSuppressing the Cell Propagation (1)

The doxorubicin-containing liposome modified with DSPE-16CC produced bythe procedure of Example 33 was evaluated for its effect of suppressingthe cell propagation using B16-BL6 mouse melanoma cell and Meth-A mousesarcoma cell.

First, amount of the phosphatidyl serine on the cell surface wasmeasured by the procedure described in Example 23. When the B16-BL6 celland the Meth-A cell were treated with FITC-labeled Annexin V andmeasured by flow cytometry, average fluorescence intensity of theB16-BL6 was 735 while the average fluorescence intensity of Meth-A cellwas 114, and the average fluorescence intensity was higher in theB16-BL6 cell.

Next, the doxorubicin-containing liposome modified with the peptidechemically modified with PEG was evaluated for its effect on suppressingthe cell propagation by inoculating the B16-BL6 cell and the Meth-A cellin 96 well plate at 1×10³ cells per well, respectively, and incubatingthe cells at 37° C. in 5% CO₂ atmosphere for 1 day. On the next day, thedoxorubicin-containing liposome modified with the DSPE-16CC was added toeach cell at a dose in concentration calculated in terms of doxorubicinof 0, 10, 30, 100, 300, 1000, and 3000 ng/mL, and the cells wereincubated at 37° C. in 5% CO₂ atmosphere for 2 days. Ten μL of WST-1solution (manufactured by TAKARA) was then added to each well for colordevelopment. The effect of suppressing the cell propagation wasevaluated by comparing the activity of each sample with the absorption(100%) of the group with no addition of the sample. It was then foundthat the doxorubicin-containing liposome wherein 2.5%, 5%, or 10% of theentire PEG molecules on the liposome corresponds to the PEG moleculeschemically bonded to the peptide had the effect of suppressing the cellpropagation expressed by IC50 value which was about 3 times at maximumhigher than that of the doxorubicin-containing liposome containing nopeptide chemically modified with PEG in the case of Meth-A cell withless expression of phosphatidyl serine, and the effect about 33 times atmaximum higher in the case of B16-BL6 cell with higher expression ofphosphatidyl serine (Table 6). TABLE 6 (PEG having the peptide of SEQ IDNO: 16 bonded thereto/total number IC₅₀ [ng/mL] of the PEG molecule) ×100 [%] B16-BL6 Meth-A 0 10000 2500 2.5 1500 2500 5 300 1500 10 350 900

Example 36 Evaluation of the Doxorubicin-Containing Liposome Modifiedwith the Peptide Chemically Modified with PEG for its Effect onSuppressing the Cell Propagation (2)

the doxorubicin-containing liposome modified with DSPE-16NC prepared bythe procedure of Example 33 was evaluated for its effect of suppressingcell propagation using B16-BL6 mouse melanoma cell by the proceduredescribed in Example 35. It was then found that thedoxorubicin-containing liposome wherein 2.5%, 5%, or 10% of the entirePEG molecules on the liposome correspond to the PEG molecules chemicallybonded to the peptide had the effect of suppressing the cell propagationexpressed by IC₅₀ value which was about 10 times at maximum higher thanthat of the doxorubicin-containing liposome containing no peptidechemically modified with PEG (Table 7). TABLE 7 (PEG having the peptideof SEQ ID NO: 16 bonded thereto/total number of the PEG molecule) × 100[%] IC₅₀ [ng/mL] 0 2000 2.5 800 5 250 10 200

Example 37 Effect of the Doxorubicin-Containing Liposome Modified withthe Peptide Chemically Modified with PEG in Extending the SurvivalPeriod of Cancer-Bearing Mice

The doxorubicin-containing liposome modified with DSPE-16NC prepared bythe procedure of Example 33 was evaluated for its effect of extendingthe survival period of a cancer-bearing mice carrying a B16-BL6mousemelanoma cell by intradermally transplanting 1.0×10⁶ B16-BL6 mousemelanoma cells in C57/BL6 mice (7 week old, female, Japan CharlesRiver), and grouping the mice 6 days after the transplantation using thetumor volume for the index. Administration in the case of control (only5% glucose solution) and the administration of liposome preparation (ata dose of 5 mg/kg calculated in terms of doxorubicin) were carried outafter 6 days and 9 days of the cell transplantation. It was then foundthat the average survival period of the cancer-bearing mouseadministered only with 5% glucose was 30.4 days, while the averagesurvival period of the cancer-bearing mouse administered with thedoxorubicin-containing liposome containing no peptide chemicallymodified with PEG was 39.2 days and the cancer-bearing mouseadministered with the doxorubicin-containing liposome wherein 6% of theentire PEG molecules on the liposome correspond to the peptidechemically modified with PEG was 48.0 days.

Comparative Example 1

A peptide (SEQ ID NO: 25) having an amino acid composition which is thesame as that of the peptide of SEQ ID NO: 1 but with an utterly randomamino acid sequence, hence a sequence which does not include “thesequence of 18 amino acids exhibiting the four sided structure of thepresent invention” was evaluated for its nucleic acid-introducingability by the procedure described in Example 9. This peptide was alsoevaluated for its affinity for phosphatidyl serine by the proceduredescribed in Example 20. The results indicate that this peptide hadneither the nucleic acid-introducing ability nor the affinity forphosphatidyl serine.

CD spectrum was also measured by the procedure described in Example 5.The results indicate that no α-helix structure is found even in thepresence of SDS.

Comparative Example 2

SEQ ID NO: 26 which was prepared by conducting amino acid substitutionin the peptide of SEQ ID NO: 1 so that the sequence does not include“the sequence of 18 amino acids exhibiting the four sided structure ofthe present invention” was evaluated for the nucleic acid-introducingability by the procedure described in Example 9. The fluorescent countwas less than 10,000, and no nucleic acid-introducing ability was found.

Comparative Example 3

Polylysine (average molecular weight: 11,000) was evaluated for itsaffinity for phosphatidyl serine by the procedure described in Example20. It was then found that polylysine had no affinity for phosphatidylserine. Polylysine was also evaluated for absorption in BSA solution bythe procedure described in Example 4. The value measured was 1 orhigher, and formation of aggregates was thus confirmed.

Comparative Example 4

46 (Niidome, T et al., J. Biol. Chem., Vol. 272, 15307 (1997)) (thepeptide of SEQ ID NO: 27) which is an amphipathic basic peptide havingα-helix structure was evaluated for its affinity for phosphatidyl serineby the procedure described in Example 20. It was then found that thispeptide had no affinity for phosphatidyl serine. This peptide was alsoevaluated for absorption in BSA solution by the procedure described inExample 4. The value measured was 1 or higher, and formation ofaggregates was thus confirmed.

Comparative Example 5

46 described in Comparative Example 4 (the peptide of SEQ ID NO: 27) wasadministered to a mouse by the procedure described in Example 26. Themouse died immediately after the administration.

It was estimated that this result reflected the situation that thispeptide is an amphipathic basic peptide having α-helix structure whicheasily forms aggregates in blood and which exhibits serious toxicityupon administration to an animal.

INDUSTRIAL APPLICABILITY OF THE INVENTION

The present invention is capable of providing a novel peptide chemicallymodified with PEG which is highly safe; which can be easily producedinto a complex with a substance which binds to the peptide (enjoyingexcellent handling convenience), the thus produced complex exhibitsexcellent solubility; which can serve a vector with high selectivite andefficient introduction of the substance which binds to the peptide; andwhose specific activity has not been compensated by the chemicalmodification with the PEG; as well as its production method.

The present invention is also capable of providing a complex of thepeptide chemically modified with PEG and a substance which binds to thepeptide, and its production method.

The present invention is also capable of providing a carrier modifiedwith the peptide chemically modified with PEG, and its productionmethod.

Typical merits of the present invention are as described below.

The peptide of the present invention is useful as a peptide vector sinceit has ability of binding to a nucleic acid and ability of introducingthe nucleic acid into a cell.

Since the peptide takes α-helix structure only in the presence of aparticular substance, it does not substantially form aggregates in serumand remains highly soluble.

In addition, when the peptide binds to a nucleic acid, the nucleic acidis stable since it is imparted with nuclease resistance.

Furthermore, the peptide has affinity for phosphatidyl serine, andtherefore, it can selectively introduce the nucleic acid to the cell,tissue, or organ at the site where the so called immune response hastaken place, for example, by inflammation, cell activation orcytotoxicity by immunocompetent cell, or apoptosis, the site where thecells have become malignantly transformed through abnormal celldivision, the site where cytotoxicity of the cells constituting bloodvessel have proceeded by the progress of blood coagulation or arterialsclerosis, the site where cytotoxic reaction has proceeded by superoxide, the site where cell activation and/or cytotoxic reaction hasproceeded by a protease, and therefore, the nucleic acid can beadministered at a reduced dose with reduced side effects.

The PEG-modified peptide of the present invention has the characters andmerits as described above, and also, because of the chemicalmodification with PEG, it exhibits improved incorporation rate into thetarget cell of the genes and the drugs, improved pharmacologicalactivity, and reduced toxicity.

1. A peptide chemically modified with polyethylene glycol (PEG),including a sequence of 18 amino acids, wherein said sequence of 18amino acids is constituted from alternately arranged two hydrophobicsides and two hydrophilic sides in α-helix structural model depicted byEdmundson wheel plots, one of said hydrophobic sides comprises 5 to 7amino acids and 80 mole % or more of this side comprises hydrophobicamino acids, one of said hydrophilic sides comprises 5 or 6 amino acids,and 80 mole % or more of this side comprises hydrophilic amino acids,and 50 mole % or more of this side comprises an amino acid selected fromthe group consisting of arginine and lysine, the other of saidhydrophobic sides comprises 2 to 4 hydrophobic amino acids, and theother of said hydrophilic sides comprises 3 to 5 amino acids and 80 mole% or more of this side comprises hydrophilic amino acids.
 2. A peptidechemically modified with PEG according to claim 1 wherein said peptidecomprises 20 or more amino acids in total; opposite ends of said peptideare N and C terminals; and any 18 consecutive amino acids in saidpeptide excluding the amino acids at opposite ends constitutes saidsequence of 18 amino acids.
 3. A peptide chemically modified with PEGaccording to claim 1 or 2 wherein the amino acids at the N and Cterminals are each a hydrophilic amino acid.
 4. A peptide chemicallymodified with PEG according to claim 1 wherein said sequence of 18 aminoacids is a sequence of any 18 consecutive amino acids in the followingamino acid sequence:X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-X32-X3 3-X34-X35-X36, provided that, in each of “X4, X8, X11, X15, andX19”, “X8, X11, X15, X19, and X22”, “X11, X15, X19, X22, and X26”, “X15,X19, X22, X26, and X29”, and “X19, X22, X26, X29, and X33”, at least 4amino acids out of the 5 amino acids are a hydrophobic amino acid, X3,X10, X12, X21, X28, and X30 are independently a hydrophobic amino acid,a neutral hydrophilic amino acid, or a basic hydrophilic amino acid, ineach of “X2, X5, X9, X13, and X16”, “X5, X9, X13, X16, and X20”, “X9,X13, X16, X20, and X23”, “X13, X16, X20, X23, and X27”, “X16, X20, X23,X27, and X31”, and “X20, X23, X27, X31, and X34”, at least 4 amino acidsout of the 5 amino acids are a neutral hydrophilic amino acid or a basichydrophilic amino acid, at least 3 amino acids of which being arginineor lysine, X6, X17, X24, and X35 are independently a hydrophobic aminoacid, and X7, X14, X18, X25, X32, and X36 are independently a neutralhydrophilic amino acid or a basic hydrophilic amino acid.
 5. A peptidechemically modified with PEG according to claim 1 wherein peptide moietyof said peptide chemically modified with PEG and including said sequenceof 18 amino acids comprises the following amino acid sequence:X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-X32-X33-X34-X35-X36-X37, provided that X1 and X37 are a hydrophilicamino acid, in each of “X4, X8, X11, X15, and X19”, “X8, X11, X15, X19,and X22”, “X11, X15, X19, X22, and X26”, “X15, X19, X22, X26, and X29”,and “X19, X22, X26, X29, and X33”, at least 4 amino acids out of the 5amino acids are a hydrophobic amino acid, X3, X10, X12, X21, X28 and X30are independently a hydrophobic amino acid, a neutral hydrophilic aminoacid, or a basic hydrophilic amino acid, in each of “X2, X5, X9, X13,and X16”, “X5, X9, X13, X16, and X20”, “X9, X13”, and “X20, X20, andX23”, “X13, X16, X20, X23, and X27”, “X16, X20, X23, X27, and X31”, and“X20, X23, X27, X31, and X34”, at least 4 amino acids out of the 5 aminoacids are a neutral hydrophilic amino acid or a basic hydrophilic aminoacid, at least 3 amino acids of which being arginine or lysine, X6, X17,X24, and X35 are independently a hydrophobic amino acid, and X7, X14,X18, X25, X32, and X36 are independently a neutral hydrophilic aminoacid or a basic hydrophilic amino acid; and wherein the sequence ofamino acids X2 to X36 may include deletion, addition, insertion, orsubstitution as long as at least 18 amino acids are conserved inconsecutive form.
 6. A peptide chemically modified with PEG according toclaim 5 wherein X1 to X37 are the following amino acids: X1 isthreonine, X37 is serine, X2, X5, X9, X20, X23, and X27 areindependently arginine or lysine, X3 and X21 are independently tyrosine,phenylalanine, serine, or arginine, X4, X17, X22, and X35 areindependently leucine, X6, X15, X24, and X33 are independently leucineor isoleucine, X7, X13, X25, and X31 are independently histidine orarginine, X8 and X26 are independently proline, X10 and X28 areindependently serine, arginine, or leucine, X11 and X29 areindependently tryptophan or leucine, X12 and X30 are independentlyvaline, leucine, or serine, X14 and X32 are independently glutamine,asparagine, or arginine, X16 and X34 are independently alanine orarginine, X18 is arginine, lysine, or serine, X19 is leucine orthreonine, and X36 is arginine or serine; and wherein the sequence ofamino acids X2 to X36 may include deletion, addition, insertion, orsubstitution as long as at least 18 amino acids are conserved inconsecutive form.
 7. A peptide chemically modified with PEG according toclaim 1 wherein peptide moiety of said peptide chemically modified withPEG and including said sequence of 18 amino acids comprises any one ofthe amino acid sequences of SEQ ID NO: 1 to SEQ ID NO:
 24. 8. A peptidechemically modified with PEG according to claim 1 wherein peptide moietyof said peptide chemically modified with PEG and including said sequenceof 18 amino acids comprises the amino acid sequence of SEQ ID NO: 16 orSEQ ID NO:
 19. 9. A peptide chemically modified with PEG according toclaim 1 wherein PEG moiety of said peptide chemically modified with PEGand including said sequence of 18 amino acids has a molecular weight ofabout 200 Da to about 100,000 Da.
 10. A complex comprising a peptidechemically modified with PEG and including a sequence of 18 amino acids,and a substance which binds to said peptide wherein said sequence of 18amino acids is constituted from alternately arranged two hydrophobicsides and two hydrophilic sides in α-helix structural model depicted byEdmundson wheel plots, one of said hydrophobic sides comprises 5 to 7amino acids and 80 mole % or more of this side comprises hydrophobicamino acids, one of said hydrophilic sides comprises 5 or 6 amino acids,and 80 mole % or more of this side comprises hydrophilic amino acids,and 50 mole % or more of this side comprises an amino acid selected fromthe group consisting of arginine and lysine, the other of saidhydrophobic sides comprises 2 to 4 hydrophobic amino acids, and theother of said hydrophilic sides comprises 3 to 5 amino acids and 80 mole% or more of this side comprises hydrophilic amino acids.
 11. A complexaccording to claim 10 wherein said peptide comprises 20 or more aminoacids in total; opposite ends of said peptide are N and C terminals; andany 18 consecutive amino acids in said peptide excluding the amino acidsat opposite ends constitutes said sequence of 18 amino acids.
 12. Acomplex according to claim 10 or 11 wherein the amino acids at the N andC terminals are each a hydrophilic amino acid.
 13. A complex accordingto claim 10 wherein said sequence of 18 amino acids is a sequence of any18 consecutive amino acids in the following amino acid sequence:X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-X32-X33-X34-X35-X36,provided that, in each of “X4, X8, X11, X15, and X19”, “X8 ,X11, X15,X19, and X22”, “X11, X15, X19, X22, and X26”, “X15, X19, X22, X26, andX29”, and “X19, X22, X26, X29, and X33”, at least 4 amino acids out ofthe 5 amino acids are a hydrophobic amino acid, X3, X10, X12, X21, X28,and X30 are independently a hydrophobic amino acid, a neutralhydrophilic amino acid, or a basic hydrophilic amino acid, in each of“X2, X5, X9, X13, and X16”, “X5, X9, X13, X16, and X20”, “X9, X13, X16,X20, and X23”, “X13, X16, X20, X23, and X27”, “X16, X20, X23, X27, andX31”, and “X20, X23, X27, X31, and X34”, at least 4 amino acids out ofthe 5 amino acids are a neutral hydrophilic amino acid or a basichydrophilic amino acid, at least 3 amino acids of which being arginineor lysine, X6, X17, X24, and X35 are independently a hydrophobic aminoacid, and X7, X14, X18, X25, X32, and X36 are independently a neutralhydrophilic amino acid or a basic hydrophilic amino acid.
 14. A complexaccording to claim 10 wherein peptide moiety of said peptide chemicallymodified with PEG and including said sequence of 18 amino acidscomprises the following amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-X25-X26-X27-X28-X29-X30-X31-X32-X33-X34-X35-X36-X37, provided that X1 and X37 are a hydrophilicamino acid, in each of “X4, X8, X11, X15, and X19”, “X8, X11, X15, X19,and X22”, “X11, X15, X19, X22, and X26”, “X15, X19, X22, X26, and X29”,and “X19, X22, X26, X29, and X33”, at least 4 amino acids out of the 5amino acids are a hydrophobic amino acid, X3, X10, X12, X21, X28 and X30are independently a hydrophobic amino acid, a neutral hydrophilic aminoacid, or a basic hydrophilic amino acid, in each of “X2, X5, X9, X13,and X16”, “X5, X9, X13, X16, and X20”, “X9, X13, X16, X20, and X23”,“X13, X16, X20, X23, and X27”, “X16, X20, X23, X27, and X31”, and “X20,X23, X27, X31, and X34”, at least 4 amino acids out of the 5 amino acidsare a neutral hydrophilic amino acid or a basic hydrophilic amino acid,at least 3 amino acids of which being arginine or lysine, X6, X17, X24,and X35 are independently a hydrophobic amino acid, and X7, X14, X18,X25, X32, and X36 are independently a neutral hydrophilic amino acid ora basic hydrophilic amino acid; and wherein the sequence of amino acidsX2 to X36 may include deletion, addition, insertion, or substitution aslong as at least 18 amino acids are conserved in consecutive form.
 15. Acomplex according to claim 14 wherein X1 to X37 are the following aminoacids: X1 is threonine, X37 is serine, X2, X5, X9, X20, X23, and X27 areindependently arginine or lysine, X3 and X21 are independently tyrosine,phenylalanine, serine, or arginine, X4, X17, X22, and X35 areindependently leucine, X6, X15, X24, and X33 are independently leucineor isoleucine, X7, X13, X25, and X31 are independently histidine orarginine, X8 and X26 are independently proline, X10 and X28 areindependently serine, arginine, or leucine, X11 and X29 areindependently tryptophan or leucine, X12 and X30 are independentlyvaline, leucine, or serine, X14 and X32 are independently glutamine,asparagine, or arginine, X16 and X34 are independently alanine orarginine, X18 is arginine, lysine, or serine, X19 is leucine orthreonine, and X36 is arginine or serine; and wherein the sequence ofamino acids X2 to X36 may include deletion, addition, insertion, orsubstitution as long as at least 18 amino acids are conserved inconsecutive form.
 16. A complex according to claim 10 wherein peptidemoiety of said peptide chemically modified with PEG and including saidsequence of 18 amino acids comprises any one of the amino acid sequencesof SEQ ID NO: 1 to SEQ ID NO:
 24. 17. A complex according to claim 10wherein peptide moiety of said peptide chemically modified with PEG andincluding said sequence of 18 amino acids comprises the amino acidsequence of SEQ ID NO: 16 or SEQ ID NO:
 19. 18. A complex according toclaim 10 wherein said substance which binds to the peptide is a nucleicacid.
 19. A complex according to claim 10 wherein PEG moiety of saidpeptide chemically modified with PEG and including said sequence of 18amino acids has a molecular weight of about 200 Da to about 100,000 Da.20. A method for producing the peptide chemically modified with PEG ofclaim 1 comprising the step of reacting a peptide comprising saidsequence of 18 amino acids with activated polyethylene glycol.
 21. Apeptide chemically modified with polyethylene glycol (PEG) which isproduced by the method of claim
 20. 22. A method for producing thecomplex of claim 10 comprising the steps of a) reacting a peptidecomprising said sequence of 18 amino acids with activated polyethyleneglycol (PEG), and b) reacting the peptide chemically modified with PEGthat is obtained in said a) with a substance which binds to saidpeptide.
 23. A method for producing the complex of claim 10 comprisingthe steps of a) reacting a peptide comprising said sequence of 18 aminoacids with a substance which binds to said peptide, and b) reacting thereaction product of said peptide and said substance which binds to saidpeptide with activated polyethylene glycol (PEG).
 24. A complex of apeptide chemically modified with polyethylene glycol (PEG) and asubstance which binds to said peptide, said complex being produced bythe method of claim 22 or
 23. 25. A carrier which is modified with thepeptide chemically modified with PEG according to claim
 1. 26. A methodfor producing the carrier of claim 25 which is modified with the peptidechemically modified with PEG comprising the steps of a) reacting apeptide comprising said sequence of 18 amino acids, or a peptidecomprising said sequence of 18 amino acids and having cysteine attachedto N or C terminal of the peptide, with activated PEG, and b) reactingthe reaction product of said a) with a carrier, or constructing acarrier by using the reaction product of said a) as a constituent.
 27. Acarrier which is modified with the peptide chemically modified with PEG,said carrier being produced by the method of claim
 26. 28. A method fordelivering a substance to the interior of a cell, said substance beingbonded to or incorporated in the carrier of claim 25 that has beenmodified with the peptide chemically modified with PEG.